Integrating LAMP-coupled Modification SPCE with SiNWs/PtNPs in an Electrochemical DNA Biosensor for Real-time Monitoring of Porcine DNA Amplification

Integrating LAMP-coupled Modification SPCE with SiNWs/PtNPs in an Electrochemical DNA Biosensor for Real-time Monitoring of Porcine DNA Amplification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrating LAMP-coupled Modification SPCE with SiNWs/PtNPs in an Electrochemical DNA Biosensor for Real-time Monitoring of Porcine DNA Amplification Norzila Kusnin, Nor Azah Yusof, Jaafar Abdullah, Suriana Sabri, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7079194/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present study describes the development and optimization of a loop-mediated isothermal amplification (LAMP) technique for the rapid and sensitive detection of porcine DNA, addressing critical needs in food safety and compliance with dietary laws such as halal and kosher. The primer sets were carefully designed to bind selectively to specific region of Sus scrofa mitochondrial DNA, thereby ensuring high specificity and amplification efficiency. The selection of LAMP was driven by its advantages, including rapid amplification time and isothermal conditions, which simplify the equipment requirements and reduce overall costs. To achieve optimal performance, the primers (F3, B3, FIP, BIP, and LF) were carefully designed to initiate strand displacement and DNA synthesis under isothermal conditions. The amplification parameters, such as temperature and incubation time, were systematically optimized, resulting in successful DNA amplification at 63°C for 60 minutes. The reaction's progress was monitored by measuring the turbidity associated with the accumulation of magnesium pyrophosphate, a reaction byproduct. Validation of the amplified products was performed using gel electrophoresis, confirming the presence of the expected DNA fragments. The amplified DNA products were subsequently detected using an advanced electrochemical DNA biosensor. This biosensor employed silicon nanowires and platinum nanoparticles (SiNWs/PtNPs) modified screen-printed carbon electrode (SPCE), with ferrocenylnaphthalene diimide (FND) serving as an intercalator for the detection of double-stranded DNA (dsDNA). The integration of LAMP with this biosensor enabled precise quantification and real-time monitoring of the DNA amplification process. The Limit of Detection (LOD) of the optimized LAMP method was determined to be 175.2 ng/µL, demonstrating its ability to detect low quantities of porcine DNA with high sensitivity. Cross-reactivity studies involving a range of meat sources and processed food matrices demonstrated the system's high reliability and specificity for detecting porcine DNA, with no false positives observed. Additionally, the biosensor effectively detected porcine DNA in binary meat mixtures, simulating real-world scenarios, and underscoring its practical applicability. Loop-mediated isothermal amplification (LAMP) Sus scrofa mtDNA Electrochemical DNA biosensor Screen-printed carbon electrode (SPCE) Porcine DNA detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Accurate identification of species-specific DNA in food items is crucial for assuring food safety, authenticity, and compliance with labelling requirements [ 1 ]. The detection of porcine DNA, in particular, holds significant importance due to dietary restrictions observed by certain religious and cultural groups, such as those adhering to halal and kosher dietary laws [ 2 ]. These restrictions strictly prohibit the consumption of pork, making the reliable identification of porcine DNA essential to avoid unintentional violations of these dietary laws. Furthermore, the presence of porcine DNA in food products raises concerns related to food adulteration and fraud, as unscrupulous manufacturers might substitute cheaper pork for more expensive meats without proper labelling, misleading consumers and potentially causing serious ethical and health implications [ 3 ]. Although polymerase chain reaction (PCR) is a highly effective method for DNA detection, it frequently necessitates sophisticated thermal cycling equipment and lengthy processing periods. In order to denature DNA, anneal primers, and extend new DNA strands, PCR needs to be heated and cooled many times. This requires advanced lab tools and trained staff. This complexity makes PCR less suitable for rapid, field-based applications where quick and accurate results are crucial, such as in food safety inspections, border controls, and on-site testing in processing plants [ 4 ]. Additionally, the need for a controlled laboratory environment limits PCR's utility in remote or resource-limited settings. To address these limitations, the establishment of a quick and accurate technique for porcine DNA detection in food products is highly desirable. Loop-Mediated Isothermal Amplification (LAMP) has gained recognition as a viable alternative, offering rapid DNA amplification under constant temperature conditions, thereby minimizing the equipment requirements and reducing the overall time needed for detection [ 5 ]. In contrast to PCR, LAMP enables amplification at a constant temperature, eliminating the need for a thermal cycler. This is achieved through the use of a DNA polymerase with strong strand displacement capability, which facilitates the efficient amplification of target DNA sequences without the need for temperature cycling. The isothermal nature of LAMP not only reduces the complexity of the required equipment but also speeds up the detection process, making it possible to obtain results within a shorter timeframe, often less than an hour. LAMP's effectiveness largely hinges on the design and optimization of its primers. The primer set generally comprises four to six oligonucleotides—including two inner primers (FIP and BIP) and two outer primers (F3 and B3)—each designed to bind to specific targets regions of the DNA sequence, thereby enhancing both specificity and detection sensitivity [ 6 ]. These primers are designed to bind six separate regions within the target DNA sequence, providing a high level of specificity. The inner primers (FIP and BIP) are particularly important as they initiate the strand displacement process, while the outer primers (F3 and B3) function to promote and accelerate the overall amplification reaction. Additional loop primers (LoopF and LoopB) can be incorporated to further enhance the speed of the reaction. The design of these primers is crucial as it directly influences the efficiency and target specificity of the LAMP reaction, determining the success of the DNA amplification and the reliability of the subsequent detection [ 7 ]. This work aimed to develop and optimize a LAMP-based technique for the sensitive detection of porcine DNA, addressing the critical need for reliable identification to ensure compliance with dietary laws and prevent food fraud. We designed and optimized a set of primers targeting specific regions of the Sus scrofa mitochondrial DNA to ensure high specificity and efficiency. Additionally, we integrated the LAMP method with an advanced electrochemical DNA biosensor based on silicon nanowires and platinum nanoparticles, enhancing detection sensitivity and enabling precise quantification of the amplified products. The performance of the LAMP-biosensor system was rigorously evaluated using various parameters, including amplification temperature and time, to achieve optimal results. The system's specificity was further validated through cross-reactivity studies with different types of meat and processed food samples, ensuring no false positives and reliable detection of porcine DNA. Additionally, we investigated the biosensor's capability to detect porcine DNA in binary meat mixtures, simulating real-world scenarios, to demonstrate its practical applicability. Materials and Methods LAMP Primer Design The PrimerExplorer V5 software ( http://primerexplorer.jp/e/ ) was applied to design the LAMP primer sets. These primers were developed based on the complete mitochondrial DNA gene of Sus scrofa, with the accession number AJ002189.1, sourced from the National Center for Biotechnology Information (NCBI) database. Five specific primer sequences, targeting an amplicon size of approximately 156 bp, were utilized for detecting porcine DNA. LAMP Reaction Condition To optimize the LAMP reaction conditions, a method outlined by Ran et al. was employed, with a few minor alterations [ 8 ]. Each reaction had a volume of 25 µL, comprising 1.6 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.8 µM LF primer, 1.8 mM dNTPs (Thermo Scientific, USA), 1x ThermoPol reaction buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton X-100 (New England Biolabs, USA), 0.6 M betaine (TCI, USA), 8 U of B st DNA polymerase (SBS Genetech Co, Beijing), and 1 µL of template DNA. Nuclease-free water was added to get the final volume to 25 µL. After a quick centrifugation, the mixture was incubated for an hour at 63°C. Turbidity due to magnesium pyrophosphate precipitation indicated successful amplification, while a lack of turbidity indicated no amplification. Each LAMP assay was performed in triplicate. Validation of LAMP Amplicon by Gel Electrophoresis To validate the results, all LAMP products were subjected to 2% (v/v) TBE agarose gel electrophoresis, enhanced with 0.1% GelRed (Biotium). The sizes of the LAMP products were determined using a 100 bp DNA ladder (1st Base) as a reference marker. Electrophoresis was conducted at 80 V for 75 minutes in a 1x TBE buffer. Gels were visualized using a UV transilluminator. Optimization of LAMP Condition The reaction temperatures were varied to 60, 63, 65, and 68°C for up to 60 minutes in order to find the perfect range for the porcine-specific LAMP assays. Three replicates (n = 3) were prepared for each temperature. Additionally, LAMP amplification times of 20, 30, 40, 50, and 60 minutes were evaluated, with each time period assessed in triplicate to ensure reliability. Food DNA Extraction Processed food samples containing pork were sourced from a local store in Malaysia, along with fresh raw meat samples including beef ( Bos taurus ), lamb ( Ovis aries ), chicken ( Gallus gallus ), and pork ( Sus scrofa ). The DNeasy mericon Food Kit from Qiagen (Germany) was utilized for DNA extraction. A NanoDropTM 2000 spectrophotometer (ThermoFisher Scientific, USA) was used to test the concentration and purity of the DNA extracted. DNA samples with A 260 /A 280 ratios between 1.8 and 2.0 were used for LAMP amplification. Electrochemical Sensor Preparation First, cyclic voltammetry was used to activate the SPCE's carbon working electrode in 0.1 M NaOH. A 4 µL suspension of silicon nanowires (SiNWs) in 2% APTES was drop-cast onto the electrode and incubated at room temperature for 24 hours. The electrode was then rinsed with 95% ethanol and baked at 70°C for 30 minutes. After cooling, 10 µL of 5 mM ethanolic 3,3'-dithiodipropionic acid (DTDPA) was applied and incubated for 2 hours at ambient temperature. Next, 10 µL of platinum nanoparticle (PtNPs) suspension was drop-cast onto the SiNWs-modified electrode. After incubating for 15 minutes at 50°C, the electrode was rinsed with deionized water and then dried with nitrogen gas. The resulting modified SPCE served as the electrochemical sensing platform for detecting LAMP-amplified porcine DNA using the FND-based biosensor system. Validation of the DNA Biosensor using LAMP Amplicon To validate the developed DNA biosensor, LAMP products derived from various meats (including raw pork, beef, lamb, chicken, canned pork, and pork sausages) were subjected to denaturation at 95°C for 10 minutes. This process converts double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), which is crucial for the DNA biosensor, as it specifically detects ssDNA. Post-denaturation, the samples were rapidly cooled on ice to prevent re-annealing. A 10 µL aliquot of denatured LAMP amplicons was applied to the surface of the biosensor and incubated at 40°C for two hours. Afterward, the biosensor was rinsed with TE buffer to remove any unbound DNA and was dried using nitrogen gas. The immobilized LAMP amplicons on the biosensor were incubated with ferrocenylnaphthalene diimide (FND) in 50 mM Tris-HCl (pH 7.6) for 20 minutes at room temperature without the application of any potential. In order to analyze the modified screen-printed carbon electrode (SPCE), differential pulse voltammetry (DPV) was conducted over a potential range of -0.5 V to 0 V. The electrode was rinsed with Tris-HCl buffer to eliminate excess FND, and the modulation amplitude was adjusted to 0.5 V. The time interval was 0.64 seconds, and the measured step potential was 0.0005 volts. Figure 1 illustrates the schematic diagram of LAMP amplicon detection. Results and Discussion LAMP Primer Design To successfully achieve LAMP amplification, it is essential to design a primer set that is both highly sensitive and specific. With the use of the PrimerExplorer V5 program ( http://primerexplorer.jp/e/ ), variables such base composition, GC concentration, and the possibility of secondary structure development were thoroughly assessed. The complete mitochondrial DNA of Sus scrofa (GenBank accession no. AJ002189.1) served as the model target for this study. Five primers that fulfilled the stringent LAMP criteria were designed to enhance the technique's efficiency, specificity, and simplicity (Table 1 ). The LAMP method utilizes a distinctive strand displacement DNA synthesis mechanism, eliminating the need for thermal cycling. This process begins with a DNA polymerase with strong strand displacement capabilities, which uses two types of specially designed primers: inner and outer. Table 1 A set of five primers was designed for LAMP, targeting the complete mitochondrial DNA of Sus scrofa (GenBank accession no. AJ002189.1). This set consists of two outer primers (F3 and B3), two inner primers (FIP and BIP), and one forward loop primer (LF) LAMP Primer DNA Sequences F3 5’GATACCCCACTATGCCTAGC-3’ B3 5’ATTGTGCTTACTATTGTTCCTT-3’ FIP 5’AAGTCCTTTGAGTTTTAGGCAGT-CCAAATAGTTACATAACA-3’ BIP 5’TAATCGATAAACCCCGATAGACCT-GGGTTTGCTGAAGATGGC-3’ LF 5’TGCGAGTAGTACTCTGGCGAAT-3’ Optimization of LAMP Parameters (Amplification Temperature and Incubation Time) Precipitate formation in the LAMP reaction is demonstrated by the subsequent reactions: (DNA) n −1 + dNTP ◊ (DNA) n + P 2 O 7 4− [ 1 ] P 2 O 7 4− + 2Mg 2+ ◊ Mg 2 P 2 O 7 [ 2 ] DNA polymerase generates pyrophosphate ions as a byproduct from dNTPs during the DNA polymerization process [ 9 ]. In the LAMP reaction buffer, these pyrophosphate ions react with magnesium ions in substantial quantities, resulting in the formation of a precipitate. Detailed chemical and spectroscopic analyses identified this precipitate as magnesium pyrophosphate. The accumulation of magnesium pyrophosphate, which causes turbidity, is directly proportional to the amount of amplified DNA produced. This turbidity serves as a visible indicator of amplicon presence, providing an optical signal that is highly useful for real-time monitoring in LAMP assays. As amplification proceeds and more DNA strands are synthesized, the turbidity of the reaction mixture visibly increases, indicating the rising number of amplicons. To enhance the efficiency and specificity of the LAMP assay, we investigated two critical parameters: amplification temperature and time. Optimizing these parameters was essential for refining the LAMP assay's performance. Reactions were carried out at different temperatures—specifically 60, 63, 65, and 68°C—to identify the optimal temperature for achieving the fastest positive reaction. Experimental results identified 63°C as the ideal temperature for the LAMP assay, yielding the maximum FND redox peak current (33.9 µA), which indicates effective hybridization at this precise temperature (Fig. 2 a and 2 b). This temperature setting not only facilitated rapid and efficient amplification but also significantly enhanced reaction kinetics when using 1 ng/µL of synthetic porcine oligonucleotide DNA as the target template. This discovery is consistent with the report by Zhang et al., which determined that 63°C is the optimal temperature for the LAMP amplification of Toxoplasma gondii DNA [ 10 ]. Similarly, Ahmed et al. employed 63°C in their LAMP reaction for DNA-H33258 to detect and differentiate meat species based on DNA analysis [ 11 ]. The effect of amplification time on the LAMP process is shown in Fig. 3 . This study investigated optimization over five distinct time intervals: 20, 30, 40, 50, and 60 minutes. The visual turbidity of the LAMP amplicons was monitored, as shown in Fig. 3 a, and the samples were subsequently quantified using a specially developed DNA biosensor. The results, presented in Fig. 3 b, indicate that the optimal amplification time is 60 minutes. This is demonstrated by the highest peak current observed, which directly correlates with the maximum level of hybridization detected in the porcine DNA samples. Although the optimal time was identified as 60 minutes, this duration is still significantly shorter and more practical compared to conventional PCR methods, which typically require 90–120 minutes and sophisticated thermal cycling equipment. LAMP offers a simplified and time-effective alternative, making it suitable for rapid field testing and point-of-need diagnostics. Table 2 compares the various techniques. Table 2 Comparison of various amplification techniques Method Amplification Time Detection Type Reference Conventional PCR 90–120 min Gel electrophoresis Standard method qPCR 60–90 min Fluorescence-based Real-time platforms Colorimetric LAMP 30–60 min Visual/turbidity Ran et al., 20168 LAMP (This study) 60 min Turbidity + Electrochemical (FND) Current work Similar optimization studies have been reported in the literature. For example, Zhang et al. optimized the amplification time for a LAMP assay targeting Toxoplasma gondii DNA, concluding that a 60-minute reaction time provided the best balance between sensitivity and specificity [ 10 ]. Their findings are consistent with our results, reinforcing the importance of optimizing reaction conditions to achieve reliable DNA amplification. Kano et al. investigated the impact of different amplification times on the performance of a LAMP assay developed for detecting white spot syndrome virus in shrimp [ 12 ]. They found that a 60-minute reaction time significantly enhanced sensitivity, which aligns with our observations that a 60-minute amplification period is optimal for porcine DNA detection. These studies collectively emphasize the importance of carefully calibrating amplification times to achieve the highest levels of accuracy and efficiency in LAMP assays. Both the amplification time and temperature optimization results highlight the practicality of using LAMP for field testing applications. The ability of LAMP to perform DNA amplification under isothermal conditions at 63°C within a relatively short 60-minute timeframe demonstrates its efficiency and simplicity. These characteristics are achieved using basic equipment, such as a water bath or a simple heating device, making LAMP a viable option for on-site testing where advanced laboratory infrastructure is unavailable [ 13 ]. DNA Extraction from Food Samples DNA was extracted from six different samples, including fresh raw meats such as pork (Sus scrofa), beef (Bos taurus), lamb (Ovis aries), and chicken (Gallus gallus). Additionally, processed foods containing pork were included, specifically canned pork and sausage. The extraction process utilized the DNeasy mericon Food Kit. Approximately 200 mg of each sample was ground using a mortar and pestle, then combined with 1 mL of Food Lysis Buffer and 2.5 µL of Proteinase K. This mixture was incubated at 60°C in a thermomixer to disrupt the structural integrity of the food samples and release DNA from protein complexes. The lysed mixture was then centrifuged for 5 minutes, and 700 µL of the clear supernatant was combined with 500 µL of chloroform. After vigorous vortexing for 15 seconds, the mixture was centrifuged for 15 minutes. A volume of 350 µL from the upper aqueous layer was combined with an equal amount of buffer PB, thoroughly vortexed, and then subjected to centrifugation for 1 minute. This mixture was applied to a QIAquick spin column containing 500 µL of Buffer AW2. A two-step centrifugation process followed to ensure the membrane was adequately dried. The purified DNA was then eluted by applying 150 µL of Buffer EB onto the column matrix. The concentration and purity of the extracted DNA were assessed with a NanoDrop™ 2000 spectrophotometer. The A 260 /A 280 ratio is a fundamental parameter in molecular biology for assessing the purity of nucleic acid preparations, particularly concerning protein contamination. This ratio is crucial for ensuring the quality of DNA samples. High quality DNA generally shows a ratio around 1.8 at A 260 /A 280 , indicating minimal protein content, whereas a ratio of approximately 2.0 suggests pure RNA samples [ 14 ]. The extracted genomic DNA from food samples demonstrated high purity, with A 260 /A 280 ratios ranging from 1.80 to 1.89, as summarized in Table 3 . Table 3 Purity of extracted DNA determined by NanoDrop™ 2000 spectrophotometer. Food sample DNA concentration (mg/mL) DNA purity (A 260 /A 280 ) Raw pork meat 0.187 1.80 Pork sausage 0.309 1.84 Canned pork 0.130 1.89 Raw chicken meat 0.089 1.89 Raw beef meat 0.430 1.84 Raw lamb meat 0.348 1.87 Mitochondrial sequences from the DNA isolated from the six samples listed above were successfully amplified using the designated loop primer pairs, and the generated amplification products were subsequently used to determine the species. The mitochondrial 12S rRNA genes were chosen as the molecular target for species identification in this study due to their suitable length for amplification and high mutation rate. Kanchanaphum et al. previously introduced simplex-LAMP methods specifically designed to target the D-loop region of Sus scrofa mitochondrial DNA [ 9 ]. Additionally, researchers have advanced the RealAmp approach with new primers, demonstrating its impressive capability to detect porcine mitochondrial DNA at levels as low as 1 pg [ 15 ]. A similar study using the LAMP approach detected as low as 0.5 pg with four pork-specific primers based on the mitochondrial DN1 gene sequence [ 8 ]. Cross-Reactivity Study using Various Food Samples Cross-reactivity studies are crucial for assessing the success of isothermal amplification. This study aimed to identify potential interference from non-target substances that might be present in the samples. Such interference can result from structural similarities, chemical interactions, or unintended binding of the assay components to substances other than the target. Figure 4 a illustrates the visual turbidity due to the formation of magnesium pyrophosphate, which correlates with the quantity of amplified DNA products. The developed biosensor was then used to quantify the LAMP amplicons. The FND peak current response of the biosensor (SPCE/SiNWs-PtNPs/ssDNA) after hybridization with various types of LAMP amplicons from raw pork meat, pork sausage, canned pork, raw beef meat, raw lamb meat, and raw poultry meat is illustrated in Fig. 4 b. Compared to the negative control (ssDNA probe), the DPV response for raw beef meat, raw lamb meat, and raw chicken meat did not exhibit any significant alterations, suggesting that no DNA hybridization occurred. This outcome is consistent with the absence of precipitation during the LAMP process. Nevertheless, the FND peak current increased when the capture DNA probe was exposed to fresh pork meat, pork sausage, and canned pork, with peak currents of 22.5 µA, 20.8 µA, and 23.9 µA, respectively. This result showed that FND has a high-affinity interaction with double-stranded DNA surfaces, resulting in a high FND redox current. The developed biosensor was only able to detect the Sus scrofa mtDNA targeted sequence. Previous studies have corroborated the specificity and reliability of LAMP assays in detecting species-specific DNA. For instance, Cho et al. successfully developed a LAMP assay to identify and discriminate eight meat species, demonstrating excellent target discrimination and detection efficiency, making it suitable for rapid diagnostic applications [ 16 ]. Similarly, Yang et al. reported a real time loop-mediated isothermal amplification (RealAmp) method for identifying of pork in meat products based on mitochondrial DNA sequences, which could differentiate between various meats (cattle, sheep, chicken and duck) with high accuracy [ 15 ]. To confirm these findings, the LAMP amplicons from all samples were further validated using agarose gel electrophoresis. As depicted in Fig. 5 , the LAMP amplification products displayed a characteristic ladder pattern, in contrast to the single discrete band commonly seen in conventional PCR. This distinctive pattern observed can be attributed to the development of branched DNA structures resembling cauliflower, caused by the annealing of inverted repeat regions from the same strand, leading to multiple loop formations. The genomic DNA extracted from the porcine samples (S1, S2, S3) was effectively amplified by the LAMP primers. Conversely, no visible bands were observed for the other species (S4, S5, S6), indicating no amplification. The presence of a positive control (C1) and the absence of bands in the negative control (C2) validated the integrity of the experimental procedure. These findings align with the electrochemical detection results, offering strong evidence of a notable molecular interaction involving the DNA probe and the complementary Sus scrofa sequences. The parallel validation through both agarose gel electrophoresis and electrochemical detection methods reinforces the robustness and accuracy of our DNA sensing approach, affirming its capability to specifically detect and interact with the target Sus scrofa DNA sequences. Validation of Developed DNA Biosensor Specificity and Capability In order to verify the specificity and efficacy of the DNA biosensor detection system that was developed, a study was conducted using binary raw meat mixtures that contained variable proportions of chicken and pork. The aim of this study was to assess the system's ability to accurately identify and amplify target DNA from mixed samples, thereby imitating real-world situations where precise species discrimination is crucial. Figure 6 illustrates the use of the designated LAMP primer combination, specifically designed for pork DNA amplification, in this study. Remarkably, the results demonstrated an exceptional level of specificity. These LAMP primers exhibited distinct behaviour when applied to binary meat mixtures with different pork-to-chicken ratios. Figure 6 a visually showcases the notable turbidity patterns observed in the binary meat mixtures, providing a clear contrast between samples containing pork and those composed entirely of chicken meat. When pork was present in the mixture, the turbidity levels showed noticeable changes, creating a visual effect that distinguished between the two scenarios. Conversely, no changes in turbidity or formation of precipitates were observed in samples consisting solely of chicken meat. The LAMP amplicons were then quantified using the developed DNA biosensor. Notably, the LAMP primers did not produce any amplification signal in the 0% pork and 100% chicken binary mixtures, which effectively supported the absence of false positives and negligible cross-reactivity with chicken DNA. The binary mixtures that contained a minimum quantity of pork DNA produced the most compelling validation, specifically at the 20% pork composition (19.9 µA) (Fig. 6 b). In this scenario, the designated LAMP primers successfully amplified the DNA within the mixture, clearly demonstrating the biosensor’s sensitivity in detecting even small traces of pork-specific target DNA. The detection of pork DNA in the mixed samples was further substantiated by the observed electrochemical responses, displayed as a significant increase in the FND redox current. This distinctive electrochemical signal serves as a direct indicator of substantial amplicon generation when pork-specific target DNA was present within the binary mixtures. While visual differences in turbidity were apparent (Fig. 6 a), the electrochemical peak current measurements in Fig. 6 b showed no significant statistical difference between the various pork-chicken mixtures. This suggests that the electrochemical method may be effective for qualitative detection (presence/absence) of pork DNA but may not precisely quantify the relative concentration of pork in mixed samples. These findings are consistent with previous studies that have utilized LAMP for species-specific DNA detection in mixed samples. For instance, Abdulmawjood et al. demonstrated the effectiveness of a LAMP assay in detecting ostrich DNA in meat products, even at low contamination levels [ 17 ]. Their research highlighted the high sensitivity and specificity of LAMP in identifying meat adulteration, similar to our results with porcine DNA detection in binary meat mixtures. Similarly, Abdullahi et al. developed a LAMP assay to detect pork DNA in mixed meat samples (pork and cattle beef), achieving reliable differentiation between target and non-target species, thereby confirming the robustness of LAMP for food authenticity testing [ 18 ]. Furthermore, the integration of the LAMP method with an electrochemical biosensor in our study parallels the work of Jaroenram et al., who employed a LAMP-electrochemical biosensor system to detect Mycobacterium tuberculosis in sputum samples [ 19 ]. Their study demonstrated the system's rapid and reliable detection capabilities, essential for timely clinical safety interventions. Our results corroborate these findings, emphasizing the practicality of using an integrated LAMP-biosensor system for on-site applications, where rapid and accurate species identification is critical. Conclusions This research reports the successful development and optimization of a Loop-Mediated Isothermal Amplification (LAMP) method for rapid and sensitive porcine DNA detection. The carefully designed primer sets, targeting specific regions of the Sus scrofa mitochondrial DNA, exhibited strong target selectivity and reliable amplification performance, positioning LAMP technique as an ideal choice for this application. The amplification parameters, including temperature and time, were meticulously optimized to ensure maximum sensitivity and reliability, with optimal results observed at 63°C over a 60-minute incubation period. Our findings indicate that the formation of a white insoluble byproduct during loop-mediated isothermal amplification serves as a dependable visual and optical signal for detecting the target DNA. The optimized LAMP method, coupled with an advanced DNA biosensor based on silicon nanowires and platinum nanoparticles, enabled precise quantification of the amplified products. The biosensor exhibited high sensitivity, detecting porcine DNA at very low concentrations, and demonstrated exceptional specificity, with no cross-reactivity observed with DNA from other meat species. The validation of the DNA biosensor using binary raw meat mixtures further underscored its capability to accurately identify and quantify pork DNA in complex samples. The distinct turbidity patterns and electrochemical responses provided clear differentiation between samples containing pork and those composed solely of chicken meat. This specificity was maintained even at low concentrations of pork DNA, highlighting the biosensor’s potential for real-world applications in food authenticity testing and species identification. Declarations Acknowledgements The authors would like to thank Universiti Putra Malaysia for their financial support [Project Number = GP-IPS/2017/9540500]. Author contributions Norzila Kusnin: conceptualization, methodology, formal analysis, investigation, writing – original draft. Nor Azah Yusof: conceptualization, investigation, supervision, writing – review & editing, funding acquisition, project administration. Jaafar Abdullah: conceptualization, investigation, supervision. Suriana Sabri: conceptualization, investigation, supervision. Shuhaimi Mustafa: conceptualization, investigation, supervision. Shinobu Sato: conceptualization, investigation, supervision. Shigeori Takenaka: conceptualization, investigation, supervision. Azizul Isha: review & editing. Funding This work was supported by the Universiti Putra Malaysia [Project Number = GP-IPS/2017/9540500]. Data Availability Data will be made available on request. Competing Interests: The authors declare no competing interests. Clinical trial number: not applicable. Animal Ethics and Consent to Participate declarations: not applicable Clinical trial number: not applicable. References Farag MDEDH (2020) Detecting adulteration in halal foods. In: Al-Teinaz YR, Spear S, Abd El-Rahim IHA (eds.) The Halal Food Handbook. pp. 283–319. https://doi.org/10.1002/9781118823026.ch18 Muhammad M, Elistina A, Ahmad SO (2020) The challenges faced by halal certification authorities in managing the halal certification process in Malaysia. Food Res 4(1):170–178. https://doi.org/10.26656/fr.2017.4(S1).S17 Bourguiba-Hachemi S, Fathallah MD (2016) DNA testing of meat foods raises issues beyond adulteration. 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Korean J Food Sci Anim Resour 34(6):799–807. https://doi.org/10.5851/kosfa.2014.34.6.799 Abdulmawjood A, Grabowski N, Fohler S, Kittler S, Nagengast H, Klein G (2014) Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PLoS ONE 9(6):e100717. https://doi.org/10.1371/journal.pone.0100717 Abdullahi U, Naim R, Wan-Taib WR, Saleh A, Muazu A, Aliyu S, Baig AA (2015) Loop-mediated isothermal amplification (LAMP), an innovation in gene amplification: bridging the gap in molecular diagnostics; a review. Ind J Sci Technol 8(17):1–12. https://doi.org/10.17485/ijst/2015/v8i17/55767 Jaroenram W, Kampeera J, Arunrut N, Karuwan C, Sappat A, Khumwan P, Jaitrong S, Boonnak K, Prammananan T, Chaiprasert A, Tuantranont A, Kiatpathomchai W (2020) Graphene-based electrochemical genosensor incorporated loop-mediated isothermal amplification for rapid on-site detection of Mycobacterium tuberculosis. J Pharm Biomed Anal 186:113333. https://doi.org/10.1016/j.jpba.2020.113333 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx UncroppedImageFig5.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7079194","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487133274,"identity":"16da4f1e-6a5e-4687-bd4d-7482aa61163f","order_by":0,"name":"Norzila Kusnin","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Norzila","middleName":"","lastName":"Kusnin","suffix":""},{"id":487133276,"identity":"63d5af5a-fd79-46f7-a7dc-6de15d26f1ab","order_by":1,"name":"Nor Azah Yusof","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYHACNgaGCghLggQtZ0jWwthGihb+/sPPHvPOO5yn28B88DYPw7bEBkJaJA4cMzfm3Xa42OwAW7I1D8NtwloYDjaYSQO1JG47wGMmTZQW+cPs36R554C08H8jTovBMaDhvA1gW9iI02J4hqdMcs6x9GKzw2zGlnMMbhsT1CJ3/vg2iTc11nlmx5sf3nhTcVuWoBYQYOJhYEhgYAa7k8GRKC2MP0BaoMCeGB2jYBSMglEwsgAA/w09YQ3Lk6MAAAAASUVORK5CYII=","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Nor","middleName":"Azah","lastName":"Yusof","suffix":""},{"id":487133277,"identity":"86262631-96d2-4d6b-b6c7-97e6499139e5","order_by":2,"name":"Jaafar Abdullah","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Jaafar","middleName":"","lastName":"Abdullah","suffix":""},{"id":487133279,"identity":"c3060f32-8470-48a4-950c-335f20e6fc21","order_by":3,"name":"Suriana Sabri","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Suriana","middleName":"","lastName":"Sabri","suffix":""},{"id":487133280,"identity":"6790f8a6-3670-4e23-a9ce-2eaad635bdb4","order_by":4,"name":"Shuhaimi Mustafa","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Shuhaimi","middleName":"","lastName":"Mustafa","suffix":""},{"id":487133281,"identity":"26ee9680-d84c-41a1-a2f7-0c1f60fa4bac","order_by":5,"name":"Shinobu Sato","email":"","orcid":"","institution":"Kyushu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shinobu","middleName":"","lastName":"Sato","suffix":""},{"id":487133282,"identity":"6588721d-99a3-4b41-af4d-cbb30a222575","order_by":6,"name":"Shigeori Takenaka","email":"","orcid":"","institution":"Kyushu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shigeori","middleName":"","lastName":"Takenaka","suffix":""},{"id":487133283,"identity":"bf24128e-7110-4137-95e8-6bc5827b075b","order_by":7,"name":"Azizul Isha","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Azizul","middleName":"","lastName":"Isha","suffix":""}],"badges":[],"createdAt":"2025-07-09 03:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7079194/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7079194/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87040161,"identity":"57a4639f-7b20-4fd3-9a50-aa42c69d8149","added_by":"auto","created_at":"2025-07-18 13:45:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1514238,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of LAMP amplicon detection using DPV method\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/fbf6410ad3cf046063fc2a0f.png"},{"id":87040163,"identity":"139c4784-3517-4a6d-9216-ca6bfb5370f1","added_by":"auto","created_at":"2025-07-18 13:45:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2521581,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different temperature for a 60-minute LAMP assay. a) Visual turbidity (the increased turbidity at 63°C indicating successful amplification) and b) Bar chart represent FND peak current of LAMP temperature in 50 mM Tris-HCl containing 1.0 M NaCl (pH 7.6) using differential pulse voltammetry (DPV) measurement (n=3)\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/6be392c6cd9357fee655fb25.png"},{"id":87040168,"identity":"978eb6d0-471e-4a56-bc91-fc9dba12d75f","added_by":"auto","created_at":"2025-07-18 13:45:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3184039,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different amplification time at 63 °C for LAMP assay. a) Visual turbidity and b) Bar chart represent FND peak current of LAMP amplification time in 50 mM Tris-HCl containing 1.0 M NaCl (pH 7.6) using differential pulse voltammetry (DPV) measurement (n=3).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/80bb2a020b813e8af43c0a39.png"},{"id":87040162,"identity":"d766a7ba-6789-4dab-b9e3-578d3e097708","added_by":"auto","created_at":"2025-07-18 13:45:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1644553,"visible":true,"origin":"","legend":"\u003cp\u003eCross-reactivity study of the developed DNA biosensor towards different food samples. a) Visual turbidity and b) Bar chart represent the FND redox peak current of food samples in 50 mM Tris-HCl containing 1.0 M NaCl (pH 7.6) at the potential range -0.5 to 0 V, step potential 0.0005 V and modulation amplitude 0.5 V (n=3)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/0c2b8ffd74fb233043eed4ca.png"},{"id":87040183,"identity":"524c9cbe-b212-4377-be3a-15c92061dfad","added_by":"auto","created_at":"2025-07-18 13:45:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1292117,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel image showing LAMP-amplified products derived from various sample types, including raw pork, pork sausage, canned pork, raw beef, raw lamb, and raw chicken (labelled S1 to S6). A synthetic porcine oligonucleotide served as the positive control (C1), while a no template reaction was used as the negative control (C2). Each reaction used 1 µg of extracted DNA. M indicated the 100 bp molecular weight marker.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/3cd2c06bef219992189e0e19.png"},{"id":87040174,"identity":"1e3996b4-23e0-469d-b95d-80a530b64509","added_by":"auto","created_at":"2025-07-18 13:45:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1365252,"visible":true,"origin":"","legend":"\u003cp\u003ea) Visual turbidity and b) Electrochemical redox peak current responses of LAMP amplicons obtained from isolated DNA of pork-chicken binary mixtures, amplified using a loop primer set specific to pork DNA. Samples 1-8 represent the following compositions: 1) 0% pork / 100% chicken, 2) 20% pork / 80% chicken, 3) 40% pork / 60% chicken, 4) 60% pork / 40% chicken, 5) 80% pork / 20% chicken, 6) 100% pork / 0% chicken, 7) 100% porcine synthetic oligonucleotide DNA (reference control), and 8) negative control (LAMP without any template DNA)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/4c23bffa96d711c833ffcac9.png"},{"id":88907060,"identity":"cbec1abc-80c1-4763-a830-459fc87958d9","added_by":"auto","created_at":"2025-08-12 14:39:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10648380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/8b2f95f4-6ba3-4772-aa2f-ea10fad28450.pdf"},{"id":87040740,"identity":"c318209f-f31f-491b-9ae4-fa58a4d98c30","added_by":"auto","created_at":"2025-07-18 13:53:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":238972,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/048caf59420f969499da7a70.docx"},{"id":87040166,"identity":"1b7bd863-c170-4cbf-aca2-b6d5cf5acd57","added_by":"auto","created_at":"2025-07-18 13:45:20","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":998956,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedImageFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7079194/v1/b30e8ab1c0499306e1c93864.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrating LAMP-coupled Modification SPCE with SiNWs/PtNPs in an Electrochemical DNA Biosensor for Real-time Monitoring of Porcine DNA Amplification","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccurate identification of species-specific DNA in food items is crucial for assuring food safety, authenticity, and compliance with labelling requirements [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The detection of porcine DNA, in particular, holds significant importance due to dietary restrictions observed by certain religious and cultural groups, such as those adhering to halal and kosher dietary laws [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These restrictions strictly prohibit the consumption of pork, making the reliable identification of porcine DNA essential to avoid unintentional violations of these dietary laws. Furthermore, the presence of porcine DNA in food products raises concerns related to food adulteration and fraud, as unscrupulous manufacturers might substitute cheaper pork for more expensive meats without proper labelling, misleading consumers and potentially causing serious ethical and health implications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although polymerase chain reaction (PCR) is a highly effective method for DNA detection, it frequently necessitates sophisticated thermal cycling equipment and lengthy processing periods. In order to denature DNA, anneal primers, and extend new DNA strands, PCR needs to be heated and cooled many times. This requires advanced lab tools and trained staff. This complexity makes PCR less suitable for rapid, field-based applications where quick and accurate results are crucial, such as in food safety inspections, border controls, and on-site testing in processing plants [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, the need for a controlled laboratory environment limits PCR's utility in remote or resource-limited settings. To address these limitations, the establishment of a quick and accurate technique for porcine DNA detection in food products is highly desirable.\u003c/p\u003e\u003cp\u003eLoop-Mediated Isothermal Amplification (LAMP) has gained recognition as a viable alternative, offering rapid DNA amplification under constant temperature conditions, thereby minimizing the equipment requirements and reducing the overall time needed for detection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast to PCR, LAMP enables amplification at a constant temperature, eliminating the need for a thermal cycler. This is achieved through the use of a DNA polymerase with strong strand displacement capability, which facilitates the efficient amplification of target DNA sequences without the need for temperature cycling. The isothermal nature of LAMP not only reduces the complexity of the required equipment but also speeds up the detection process, making it possible to obtain results within a shorter timeframe, often less than an hour. LAMP's effectiveness largely hinges on the design and optimization of its primers. The primer set generally comprises four to six oligonucleotides\u0026mdash;including two inner primers (FIP and BIP) and two outer primers (F3 and B3)\u0026mdash;each designed to bind to specific targets regions of the DNA sequence, thereby enhancing both specificity and detection sensitivity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These primers are designed to bind six separate regions within the target DNA sequence, providing a high level of specificity. The inner primers (FIP and BIP) are particularly important as they initiate the strand displacement process, while the outer primers (F3 and B3) function to promote and accelerate the overall amplification reaction. Additional loop primers (LoopF and LoopB) can be incorporated to further enhance the speed of the reaction. The design of these primers is crucial as it directly influences the efficiency and target specificity of the LAMP reaction, determining the success of the DNA amplification and the reliability of the subsequent detection [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e This work aimed to develop and optimize a LAMP-based technique for the sensitive detection of porcine DNA, addressing the critical need for reliable identification to ensure compliance with dietary laws and prevent food fraud. We designed and optimized a set of primers targeting specific regions of the \u003cem\u003eSus scrofa\u003c/em\u003e mitochondrial DNA to ensure high specificity and efficiency. Additionally, we integrated the LAMP method with an advanced electrochemical DNA biosensor based on silicon nanowires and platinum nanoparticles, enhancing detection sensitivity and enabling precise quantification of the amplified products. The performance of the LAMP-biosensor system was rigorously evaluated using various parameters, including amplification temperature and time, to achieve optimal results. The system's specificity was further validated through cross-reactivity studies with different types of meat and processed food samples, ensuring no false positives and reliable detection of porcine DNA. Additionally, we investigated the biosensor's capability to detect porcine DNA in binary meat mixtures, simulating real-world scenarios, to demonstrate its practical applicability.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eLAMP Primer Design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe PrimerExplorer V5 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://primerexplorer.jp/e/\u003c/span\u003e\u003cspan address=\"http://primerexplorer.jp/e/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was applied to design the LAMP primer sets. These primers were developed based on the complete mitochondrial DNA gene of Sus scrofa, with the accession number AJ002189.1, sourced from the National Center for Biotechnology Information (NCBI) database. Five specific primer sequences, targeting an amplicon size of approximately 156 bp, were utilized for detecting porcine DNA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLAMP Reaction Condition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo optimize the LAMP reaction conditions, a method outlined by Ran et al. was employed, with a few minor alterations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Each reaction had a volume of 25 \u0026micro;L, comprising 1.6 \u0026micro;M each of FIP and BIP, 0.2 \u0026micro;M each of F3 and B3, 0.8 \u0026micro;M LF primer, 1.8 mM dNTPs (Thermo Scientific, USA), 1x ThermoPol reaction buffer (20 mM Tris-HCl, 10 mM (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 10 mM KCl, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.1% Triton X-100 (New England Biolabs, USA), 0.6 M betaine (TCI, USA), 8 U of B\u003cem\u003est\u003c/em\u003e DNA polymerase (SBS Genetech Co, Beijing), and 1 \u0026micro;L of template DNA. Nuclease-free water was added to get the final volume to 25 \u0026micro;L. After a quick centrifugation, the mixture was incubated for an hour at 63\u0026deg;C. Turbidity due to magnesium pyrophosphate precipitation indicated successful amplification, while a lack of turbidity indicated no amplification. Each LAMP assay was performed in triplicate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of LAMP Amplicon by Gel Electrophoresis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo validate the results, all LAMP products were subjected to 2% (v/v) TBE agarose gel electrophoresis, enhanced with 0.1% GelRed (Biotium). The sizes of the LAMP products were determined using a 100 bp DNA ladder (1st Base) as a reference marker. Electrophoresis was conducted at 80 V for 75 minutes in a 1x TBE buffer. Gels were visualized using a UV transilluminator.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptimization of LAMP Condition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe reaction temperatures were varied to 60, 63, 65, and 68\u0026deg;C for up to 60 minutes in order to find the perfect range for the porcine-specific LAMP assays. Three replicates (n\u0026thinsp;=\u0026thinsp;3) were prepared for each temperature. Additionally, LAMP amplification times of 20, 30, 40, 50, and 60 minutes were evaluated, with each time period assessed in triplicate to ensure reliability.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFood DNA Extraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProcessed food samples containing pork were sourced from a local store in Malaysia, along with fresh raw meat samples including beef (\u003cem\u003eBos taurus\u003c/em\u003e), lamb (\u003cem\u003eOvis aries\u003c/em\u003e), chicken (\u003cem\u003eGallus gallus\u003c/em\u003e), and pork (\u003cem\u003eSus scrofa\u003c/em\u003e). The DNeasy mericon Food Kit from Qiagen (Germany) was utilized for DNA extraction. A NanoDropTM 2000 spectrophotometer (ThermoFisher Scientific, USA) was used to test the concentration and purity of the DNA extracted. DNA samples with A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e ratios between 1.8 and 2.0 were used for LAMP amplification.\u003c/p\u003e\u003cp\u003e\u003cb\u003eElectrochemical Sensor Preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFirst, cyclic voltammetry was used to activate the SPCE's carbon working electrode in 0.1 M NaOH. A 4 \u0026micro;L suspension of silicon nanowires (SiNWs) in 2% APTES was drop-cast onto the electrode and incubated at room temperature for 24 hours. The electrode was then rinsed with 95% ethanol and baked at 70\u0026deg;C for 30 minutes. After cooling, 10 \u0026micro;L of 5 mM ethanolic 3,3'-dithiodipropionic acid (DTDPA) was applied and incubated for 2 hours at ambient temperature. Next, 10 \u0026micro;L of platinum nanoparticle (PtNPs) suspension was drop-cast onto the SiNWs-modified electrode. After incubating for 15 minutes at 50\u0026deg;C, the electrode was rinsed with deionized water and then dried with nitrogen gas. The resulting modified SPCE served as the electrochemical sensing platform for detecting LAMP-amplified porcine DNA using the FND-based biosensor system.\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of the DNA Biosensor using LAMP Amplicon\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo validate the developed DNA biosensor, LAMP products derived from various meats (including raw pork, beef, lamb, chicken, canned pork, and pork sausages) were subjected to denaturation at 95\u0026deg;C for 10 minutes. This process converts double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), which is crucial for the DNA biosensor, as it specifically detects ssDNA. Post-denaturation, the samples were rapidly cooled on ice to prevent re-annealing. A 10 \u0026micro;L aliquot of denatured LAMP amplicons was applied to the surface of the biosensor and incubated at 40\u0026deg;C for two hours. Afterward, the biosensor was rinsed with TE buffer to remove any unbound DNA and was dried using nitrogen gas. The immobilized LAMP amplicons on the biosensor were incubated with ferrocenylnaphthalene diimide (FND) in 50 mM Tris-HCl (pH 7.6) for 20 minutes at room temperature without the application of any potential. In order to analyze the modified screen-printed carbon electrode (SPCE), differential pulse voltammetry (DPV) was conducted over a potential range of -0.5 V to 0 V. The electrode was rinsed with Tris-HCl buffer to eliminate excess FND, and the modulation amplitude was adjusted to 0.5 V. The time interval was 0.64 seconds, and the measured step potential was 0.0005 volts. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the schematic diagram of LAMP amplicon detection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003eLAMP Primer Design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo successfully achieve LAMP amplification, it is essential to design a primer set that is both highly sensitive and specific. With the use of the PrimerExplorer V5 program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://primerexplorer.jp/e/\u003c/span\u003e\u003cspan address=\"http://primerexplorer.jp/e/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), variables such base composition, GC concentration, and the possibility of secondary structure development were thoroughly assessed. The complete mitochondrial DNA of \u003cem\u003eSus scrofa\u003c/em\u003e (GenBank accession no. AJ002189.1) served as the model target for this study. Five primers that fulfilled the stringent LAMP criteria were designed to enhance the technique's efficiency, specificity, and simplicity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The LAMP method utilizes a distinctive strand displacement DNA synthesis mechanism, eliminating the need for thermal cycling. This process begins with a DNA polymerase with strong strand displacement capabilities, which uses two types of specially designed primers: inner and outer.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eA set of five primers was designed for LAMP, targeting the complete mitochondrial DNA of Sus scrofa (GenBank accession no. AJ002189.1). This set consists of two outer primers (F3 and B3), two inner primers (FIP and BIP), and one forward loop primer (LF)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLAMP Primer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDNA Sequences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;GATACCCCACTATGCCTAGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;ATTGTGCTTACTATTGTTCCTT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;AAGTCCTTTGAGTTTTAGGCAGT-CCAAATAGTTACATAACA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;TAATCGATAAACCCCGATAGACCT-GGGTTTGCTGAAGATGGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;TGCGAGTAGTACTCTGGCGAAT-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptimization of LAMP Parameters (Amplification Temperature and Incubation Time)\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrecipitate formation in the LAMP reaction is demonstrated by the subsequent reactions:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e(DNA)\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u0026minus;1\u003c/sub\u003e + dNTP \u0026loz; (DNA)\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e + P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003csup\u003e4\u0026minus;\u003c/sup\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003csup\u003e4\u0026minus;\u003c/sup\u003e + 2Mg\u003csup\u003e2+\u003c/sup\u003e \u0026loz; Mg\u003csub\u003e2\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDNA polymerase generates pyrophosphate ions as a byproduct from dNTPs during the DNA polymerization process [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the LAMP reaction buffer, these pyrophosphate ions react with magnesium ions in substantial quantities, resulting in the formation of a precipitate. Detailed chemical and spectroscopic analyses identified this precipitate as magnesium pyrophosphate. The accumulation of magnesium pyrophosphate, which causes turbidity, is directly proportional to the amount of amplified DNA produced. This turbidity serves as a visible indicator of amplicon presence, providing an optical signal that is highly useful for real-time monitoring in LAMP assays. As amplification proceeds and more DNA strands are synthesized, the turbidity of the reaction mixture visibly increases, indicating the rising number of amplicons.\u003c/p\u003e\u003cp\u003eTo enhance the efficiency and specificity of the LAMP assay, we investigated two critical parameters: amplification temperature and time. Optimizing these parameters was essential for refining the LAMP assay's performance. Reactions were carried out at different temperatures\u0026mdash;specifically 60, 63, 65, and 68\u0026deg;C\u0026mdash;to identify the optimal temperature for achieving the fastest positive reaction. Experimental results identified 63\u0026deg;C as the ideal temperature for the LAMP assay, yielding the maximum FND redox peak current (33.9 \u0026micro;A), which indicates effective hybridization at this precise temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This temperature setting not only facilitated rapid and efficient amplification but also significantly enhanced reaction kinetics when using 1 ng/\u0026micro;L of synthetic porcine oligonucleotide DNA as the target template. This discovery is consistent with the report by Zhang et al., which determined that 63\u0026deg;C is the optimal temperature for the LAMP amplification of Toxoplasma gondii DNA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similarly, Ahmed et al. employed 63\u0026deg;C in their LAMP reaction for DNA-H33258 to detect and differentiate meat species based on DNA analysis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effect of amplification time on the LAMP process is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. This study investigated optimization over five distinct time intervals: 20, 30, 40, 50, and 60 minutes. The visual turbidity of the LAMP amplicons was monitored, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, and the samples were subsequently quantified using a specially developed DNA biosensor. The results, presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, indicate that the optimal amplification time is 60 minutes. This is demonstrated by the highest peak current observed, which directly correlates with the maximum level of hybridization detected in the porcine DNA samples. Although the optimal time was identified as 60 minutes, this duration is still significantly shorter and more practical compared to conventional PCR methods, which typically require 90\u0026ndash;120 minutes and sophisticated thermal cycling equipment. LAMP offers a simplified and time-effective alternative, making it suitable for rapid field testing and point-of-need diagnostics. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e compares the various techniques.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of various amplification techniques\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmplification Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDetection Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConventional PCR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90\u0026ndash;120 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGel electrophoresis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStandard method\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eqPCR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u0026ndash;90 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFluorescence-based\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReal-time platforms\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColorimetric LAMP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u0026ndash;60 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVisual/turbidity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRan et al., 20168\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLAMP (This study)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTurbidity\u0026thinsp;+\u0026thinsp;Electrochemical (FND)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCurrent work\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSimilar optimization studies have been reported in the literature. For example, Zhang et al. optimized the amplification time for a LAMP assay targeting \u003cem\u003eToxoplasma gondii\u003c/em\u003e DNA, concluding that a 60-minute reaction time provided the best balance between sensitivity and specificity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Their findings are consistent with our results, reinforcing the importance of optimizing reaction conditions to achieve reliable DNA amplification.\u003c/p\u003e\u003cp\u003eKano et al. investigated the impact of different amplification times on the performance of a LAMP assay developed for detecting white spot syndrome virus in shrimp [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They found that a 60-minute reaction time significantly enhanced sensitivity, which aligns with our observations that a 60-minute amplification period is optimal for porcine DNA detection. These studies collectively emphasize the importance of carefully calibrating amplification times to achieve the highest levels of accuracy and efficiency in LAMP assays.\u003c/p\u003e\u003cp\u003eBoth the amplification time and temperature optimization results highlight the practicality of using LAMP for field testing applications. The ability of LAMP to perform DNA amplification under isothermal conditions at 63\u0026deg;C within a relatively short 60-minute timeframe demonstrates its efficiency and simplicity. These characteristics are achieved using basic equipment, such as a water bath or a simple heating device, making LAMP a viable option for on-site testing where advanced laboratory infrastructure is unavailable [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA Extraction from Food Samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDNA was extracted from six different samples, including fresh raw meats such as pork (Sus scrofa), beef (Bos taurus), lamb (Ovis aries), and chicken (Gallus gallus). Additionally, processed foods containing pork were included, specifically canned pork and sausage. The extraction process utilized the DNeasy mericon Food Kit. Approximately 200 mg of each sample was ground using a mortar and pestle, then combined with 1 mL of Food Lysis Buffer and 2.5 \u0026micro;L of Proteinase K. This mixture was incubated at 60\u0026deg;C in a thermomixer to disrupt the structural integrity of the food samples and release DNA from protein complexes. The lysed mixture was then centrifuged for 5 minutes, and 700 \u0026micro;L of the clear supernatant was combined with 500 \u0026micro;L of chloroform. After vigorous vortexing for 15 seconds, the mixture was centrifuged for 15 minutes. A volume of 350 \u0026micro;L from the upper aqueous layer was combined with an equal amount of buffer PB, thoroughly vortexed, and then subjected to centrifugation for 1 minute. This mixture was applied to a QIAquick spin column containing 500 \u0026micro;L of Buffer AW2. A two-step centrifugation process followed to ensure the membrane was adequately dried. The purified DNA was then eluted by applying 150 \u0026micro;L of Buffer EB onto the column matrix. The concentration and purity of the extracted DNA were assessed with a NanoDrop\u0026trade; 2000 spectrophotometer.\u003c/p\u003e\u003cp\u003eThe A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e ratio is a fundamental parameter in molecular biology for assessing the purity of nucleic acid preparations, particularly concerning protein contamination. This ratio is crucial for ensuring the quality of DNA samples. High quality DNA generally shows a ratio around 1.8 at A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e, indicating minimal protein content, whereas a ratio of approximately 2.0 suggests pure RNA samples [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The extracted genomic DNA from food samples demonstrated high purity, with A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e ratios ranging from 1.80 to 1.89, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePurity of extracted DNA determined by NanoDrop\u0026trade; 2000 spectrophotometer.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFood sample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDNA concentration (mg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDNA purity (A\u003csub\u003e260\u003c/sub\u003e/A\u003csub\u003e280\u003c/sub\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRaw pork meat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePork sausage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.309\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCanned pork\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRaw chicken meat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRaw beef meat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.430\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRaw lamb meat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMitochondrial sequences from the DNA isolated from the six samples listed above were successfully amplified using the designated loop primer pairs, and the generated amplification products were subsequently used to determine the species. The mitochondrial 12S rRNA genes were chosen as the molecular target for species identification in this study due to their suitable length for amplification and high mutation rate. Kanchanaphum et al. previously introduced simplex-LAMP methods specifically designed to target the D-loop region of \u003cem\u003eSus scrofa\u003c/em\u003e mitochondrial DNA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, researchers have advanced the RealAmp approach with new primers, demonstrating its impressive capability to detect porcine mitochondrial DNA at levels as low as 1 pg [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A similar study using the LAMP approach detected as low as 0.5 pg with four pork-specific primers based on the mitochondrial DN1 gene sequence [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eCross-Reactivity Study using Various Food Samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCross-reactivity studies are crucial for assessing the success of isothermal amplification. This study aimed to identify potential interference from non-target substances that might be present in the samples. Such interference can result from structural similarities, chemical interactions, or unintended binding of the assay components to substances other than the target. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea illustrates the visual turbidity due to the formation of magnesium pyrophosphate, which correlates with the quantity of amplified DNA products.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe developed biosensor was then used to quantify the LAMP amplicons. The FND peak current response of the biosensor (SPCE/SiNWs-PtNPs/ssDNA) after hybridization with various types of LAMP amplicons from raw pork meat, pork sausage, canned pork, raw beef meat, raw lamb meat, and raw poultry meat is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. Compared to the negative control (ssDNA probe), the DPV response for raw beef meat, raw lamb meat, and raw chicken meat did not exhibit any significant alterations, suggesting that no DNA hybridization occurred. This outcome is consistent with the absence of precipitation during the LAMP process. Nevertheless, the FND peak current increased when the capture DNA probe was exposed to fresh pork meat, pork sausage, and canned pork, with peak currents of 22.5 \u0026micro;A, 20.8 \u0026micro;A, and 23.9 \u0026micro;A, respectively. This result showed that FND has a high-affinity interaction with double-stranded DNA surfaces, resulting in a high FND redox current. The developed biosensor was only able to detect the Sus scrofa mtDNA targeted sequence.\u003c/p\u003e\u003cp\u003ePrevious studies have corroborated the specificity and reliability of LAMP assays in detecting species-specific DNA. For instance, Cho et al. successfully developed a LAMP assay to identify and discriminate eight meat species, demonstrating excellent target discrimination and detection efficiency, making it suitable for rapid diagnostic applications [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, Yang et al. reported a real time loop-mediated isothermal amplification (RealAmp) method for identifying of pork in meat products based on mitochondrial DNA sequences, which could differentiate between various meats (cattle, sheep, chicken and duck) with high accuracy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo confirm these findings, the LAMP amplicons from all samples were further validated using agarose gel electrophoresis. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the LAMP amplification products displayed a characteristic ladder pattern, in contrast to the single discrete band commonly seen in conventional PCR. This distinctive pattern observed can be attributed to the development of branched DNA structures resembling cauliflower, caused by the annealing of inverted repeat regions from the same strand, leading to multiple loop formations. The genomic DNA extracted from the porcine samples (S1, S2, S3) was effectively amplified by the LAMP primers. Conversely, no visible bands were observed for the other species (S4, S5, S6), indicating no amplification. The presence of a positive control (C1) and the absence of bands in the negative control (C2) validated the integrity of the experimental procedure. These findings align with the electrochemical detection results, offering strong evidence of a notable molecular interaction involving the DNA probe and the complementary \u003cem\u003eSus scrofa\u003c/em\u003e sequences. The parallel validation through both agarose gel electrophoresis and electrochemical detection methods reinforces the robustness and accuracy of our DNA sensing approach, affirming its capability to specifically detect and interact with the target \u003cem\u003eSus scrofa\u003c/em\u003e DNA sequences.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of Developed DNA Biosensor Specificity and Capability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn order to verify the specificity and efficacy of the DNA biosensor detection system that was developed, a study was conducted using binary raw meat mixtures that contained variable proportions of chicken and pork. The aim of this study was to assess the system's ability to accurately identify and amplify target DNA from mixed samples, thereby imitating real-world situations where precise species discrimination is crucial.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the use of the designated LAMP primer combination, specifically designed for pork DNA amplification, in this study. Remarkably, the results demonstrated an exceptional level of specificity. These LAMP primers exhibited distinct behaviour when applied to binary meat mixtures with different pork-to-chicken ratios. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea visually showcases the notable turbidity patterns observed in the binary meat mixtures, providing a clear contrast between samples containing pork and those composed entirely of chicken meat. When pork was present in the mixture, the turbidity levels showed noticeable changes, creating a visual effect that distinguished between the two scenarios. Conversely, no changes in turbidity or formation of precipitates were observed in samples consisting solely of chicken meat.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe LAMP amplicons were then quantified using the developed DNA biosensor. Notably, the LAMP primers did not produce any amplification signal in the 0% pork and 100% chicken binary mixtures, which effectively supported the absence of false positives and negligible cross-reactivity with chicken DNA. The binary mixtures that contained a minimum quantity of pork DNA produced the most compelling validation, specifically at the 20% pork composition (19.9 \u0026micro;A) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eIn this scenario, the designated LAMP primers successfully amplified the DNA within the mixture, clearly demonstrating the biosensor\u0026rsquo;s sensitivity in detecting even small traces of pork-specific target DNA. The detection of pork DNA in the mixed samples was further substantiated by the observed electrochemical responses, displayed as a significant increase in the FND redox current. This distinctive electrochemical signal serves as a direct indicator of substantial amplicon generation when pork-specific target DNA was present within the binary mixtures. While visual differences in turbidity were apparent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), the electrochemical peak current measurements in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb showed no significant statistical difference between the various pork-chicken mixtures. This suggests that the electrochemical method may be effective for qualitative detection (presence/absence) of pork DNA but may not precisely quantify the relative concentration of pork in mixed samples.\u003c/p\u003e\u003cp\u003eThese findings are consistent with previous studies that have utilized LAMP for species-specific DNA detection in mixed samples. For instance, Abdulmawjood et al. demonstrated the effectiveness of a LAMP assay in detecting ostrich DNA in meat products, even at low contamination levels [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Their research highlighted the high sensitivity and specificity of LAMP in identifying meat adulteration, similar to our results with porcine DNA detection in binary meat mixtures. Similarly, Abdullahi et al. developed a LAMP assay to detect pork DNA in mixed meat samples (pork and cattle beef), achieving reliable differentiation between target and non-target species, thereby confirming the robustness of LAMP for food authenticity testing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, the integration of the LAMP method with an electrochemical biosensor in our study parallels the work of Jaroenram et al., who employed a LAMP-electrochemical biosensor system to detect \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e in sputum samples [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Their study demonstrated the system's rapid and reliable detection capabilities, essential for timely clinical safety interventions. Our results corroborate these findings, emphasizing the practicality of using an integrated LAMP-biosensor system for on-site applications, where rapid and accurate species identification is critical.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis research reports the successful development and optimization of a Loop-Mediated Isothermal Amplification (LAMP) method for rapid and sensitive porcine DNA detection. The carefully designed primer sets, targeting specific regions of the \u003cem\u003eSus scrofa\u003c/em\u003e mitochondrial DNA, exhibited strong target selectivity and reliable amplification performance, positioning LAMP technique as an ideal choice for this application. The amplification parameters, including temperature and time, were meticulously optimized to ensure maximum sensitivity and reliability, with optimal results observed at 63\u0026deg;C over a 60-minute incubation period. Our findings indicate that the formation of a white insoluble byproduct during loop-mediated isothermal amplification serves as a dependable visual and optical signal for detecting the target DNA. The optimized LAMP method, coupled with an advanced DNA biosensor based on silicon nanowires and platinum nanoparticles, enabled precise quantification of the amplified products. The biosensor exhibited high sensitivity, detecting porcine DNA at very low concentrations, and demonstrated exceptional specificity, with no cross-reactivity observed with DNA from other meat species. The validation of the DNA biosensor using binary raw meat mixtures further underscored its capability to accurately identify and quantify pork DNA in complex samples. The distinct turbidity patterns and electrochemical responses provided clear differentiation between samples containing pork and those composed solely of chicken meat. This specificity was maintained even at low concentrations of pork DNA, highlighting the biosensor\u0026rsquo;s potential for real-world applications in food authenticity testing and species identification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThe authors would like to thank Universiti Putra Malaysia for their financial support [Project Number = GP-IPS/2017/9540500].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eNorzila Kusnin: conceptualization, methodology, formal analysis, investigation, writing \u0026ndash; original draft. Nor Azah Yusof: conceptualization, investigation, supervision, writing \u0026ndash; review \u0026amp; editing, funding acquisition, project administration. Jaafar Abdullah: conceptualization, investigation, supervision. Suriana Sabri: conceptualization, investigation, supervision. Shuhaimi Mustafa: conceptualization, investigation, supervision. Shinobu Sato: conceptualization, investigation, supervision. Shigeori Takenaka: conceptualization, investigation, supervision. Azizul Isha: review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by the Universiti Putra Malaysia [Project Number = GP-IPS/2017/9540500].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e Data will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics and Consent to Participate declarations:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFarag MDEDH (2020) Detecting adulteration in halal foods. 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J Pharm Biomed Anal 186:113333. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jpba.2020.113333\u003c/span\u003e\u003cspan address=\"10.1016/j.jpba.2020.113333\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Loop-mediated isothermal amplification (LAMP), Sus scrofa mtDNA, Electrochemical DNA biosensor, Screen-printed carbon electrode (SPCE), Porcine DNA detection","lastPublishedDoi":"10.21203/rs.3.rs-7079194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7079194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study describes the development and optimization of a loop-mediated isothermal amplification (LAMP) technique for the rapid and sensitive detection of porcine DNA, addressing critical needs in food safety and compliance with dietary laws such as halal and kosher. The primer sets were carefully designed to bind selectively to specific region of \u003cem\u003eSus scrofa\u003c/em\u003e mitochondrial DNA, thereby ensuring high specificity and amplification efficiency. The selection of LAMP was driven by its advantages, including rapid amplification time and isothermal conditions, which simplify the equipment requirements and reduce overall costs. To achieve optimal performance, the primers (F3, B3, FIP, BIP, and LF) were carefully designed to initiate strand displacement and DNA synthesis under isothermal conditions. The amplification parameters, such as temperature and incubation time, were systematically optimized, resulting in successful DNA amplification at 63\u0026deg;C for 60 minutes. The reaction's progress was monitored by measuring the turbidity associated with the accumulation of magnesium pyrophosphate, a reaction byproduct. Validation of the amplified products was performed using gel electrophoresis, confirming the presence of the expected DNA fragments. The amplified DNA products were subsequently detected using an advanced electrochemical DNA biosensor. This biosensor employed silicon nanowires and platinum nanoparticles (SiNWs/PtNPs) modified screen-printed carbon electrode (SPCE), with ferrocenylnaphthalene diimide (FND) serving as an intercalator for the detection of double-stranded DNA (dsDNA). The integration of LAMP with this biosensor enabled precise quantification and real-time monitoring of the DNA amplification process. The Limit of Detection (LOD) of the optimized LAMP method was determined to be 175.2 ng/\u0026micro;L, demonstrating its ability to detect low quantities of porcine DNA with high sensitivity. Cross-reactivity studies involving a range of meat sources and processed food matrices demonstrated the system's high reliability and specificity for detecting porcine DNA, with no false positives observed. Additionally, the biosensor effectively detected porcine DNA in binary meat mixtures, simulating real-world scenarios, and underscoring its practical applicability.\u003c/p\u003e","manuscriptTitle":"Integrating LAMP-coupled Modification SPCE with SiNWs/PtNPs in an Electrochemical DNA Biosensor for Real-time Monitoring of Porcine DNA Amplification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 13:45:16","doi":"10.21203/rs.3.rs-7079194/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8520680e-49b6-4067-9390-31b16a1c974d","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T14:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 13:45:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7079194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7079194","identity":"rs-7079194","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}