Enhancing Food Authenticity: A Dye-Based Polymerase Spiral Reaction for Rapid Detection of Duck Tissue as Adulterants in Meat Samples

Enhancing Food Authenticity: A Dye-Based Polymerase Spiral Reaction for Rapid Detection of Duck Tissue as Adulterants in Meat Samples | 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 Enhancing Food Authenticity: A Dye-Based Polymerase Spiral Reaction for Rapid Detection of Duck Tissue as Adulterants in Meat Samples Ranjita Chatterjee, Sourabh Sulabh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8627709/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Meat adulteration is a significant global concern for the food industry, compromising food safety by substituting higher-value meat with lower-cost alternatives. Duck meat is frequently used to adulterate other, more expensive commercial meats due to its similar texture and appearance. This study aimed to develop a polymerase spiral reaction (PSR)-based assay for the rapid detection of duck meat in meat samples of other species adulterated with duck tissue. The 12S rRNA duck gene was targeted for PSR-based amplification. The optimized assay was completed in 60 min when the temperature was maintained at 65°C, resulting in the successful detection of Duck DNA with a sensitivity of up to 3 fg/µL of genomic DNA. The study showed no cross-reactivity with eight other meat species and detected up to 0.01% duck adulteration in sheep meat admixtures. The PSR assay was able to precisely determine the duck content in 30 heat-treated and 20 frozen/processed samples, thus demonstrating the robustness of the developed assay. This PSR-based detection method using 12S rRNA can provide a rapid, sensitive, and cost-effective approach for the on-site detection of duck meat adulteration. Adulteration Duck meat on-site detection PSR assay rapid detection 12S rRNA gene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Meat and meat products play an important role in a balanced meat-based diet and significantly contribute to the necessary protein intake in the daily diet. Meat adulteration has become an important issue as it affects food safety, consumer trust, and market regulation worldwide (Wang et al., 2023 ). Despite all food safety laws and protocols, fraud in meat and meat products has the highest number of cases among all types (Marvin et al., 2022 ). According to market surveys in Greece, the U.S, Canada, and China, this malpractice is a crucial global scenario (Stamatis et al., 2015 ; Kane and Hellberg, 2016 ; Shehata et al., 2019 ; Song et al., 2019 ). This type of adulteration in meat and meat products may not only cause economic loss to consumers but also lead to severe health problems, raising health concerns. Duck meat is frequently mixed as an adulterant in other commercial meats, such as beef, lamb, or chicken, because of its low cost and similar texture and appearance (Zhou et al., 2023 ). In China, butter, mutton, or beef essence is used to cover up the duck odor, especially in processed items such as skewers or sausages, so that they can be easily substituted for authentic meat or meat products (Chen et al., 2022 ). The Mislabelling of meat products when the most common adulterant was duck meat has also been reported (Song et al., 2019 ). This is a clear case of economic fraud as consumers pay higher prices for cheaper meat. In studies, it has been found that individuals with avian meat allergies, such as “bird–egg syndrome, and persons reacting to one bird species' meat (e.g., chicken) can also react to duck meat, probably due to the presence of similar cross-reacting proteins having binding capabilities with IgE antibodies (Kelso et al., 1999 ). This reaction can lead to skin rashes, itching, respiratory problems, or even anaphylaxis in severe cases (Wanniang et al., 2022 ). Histamine accumulation in the consumer's body causes the main clinical symptoms such as itching, swelling, headaches, gastrointestinal issues, and bronchospasm (Lisiecka, M. Z., 2025). Several methods, such as chromatography (Di Stefano et al., 2012 ) and spectroscopy (Weng et al., 2020 ), have been used to detect meat adulteration; however, their use in the food industry is very limited. However, several protein- and DNA-based methods (He et al., 2020 ) have emerged as crucial techniques for rapid and accurate detection. Enzyme-linked immunosorbent assay (ELISA) is a less time-consuming and easy-to-perform method; however, its ability to distinguish between closely related species is unsatisfactory, and it is less effective in checking the authenticity of processed meat products as the target protein denatures during harsh processing conditions. Polymerase Chain Reaction (PCR) is a DNA-based method that detects meat adulteration more rapidly and with better accuracy; in particular, real-time PCR is more efficient for detecting processed meat products as DNA is more thermostable. However, the entire PCR procedure is performed under precise temperature control with specialized equipment, making it inconvenient to perform without a proper laboratory setup and not a great choice for point-of-care testing (POCT) (Perestam et al., 2017 ). Nucleic acid-based isothermal amplification techniques are categorized based on various reaction principles and are used for the detection of meat adulteration. According to previous studies, LAMP, SEA, CPA, and RPA have been the four most frequently utilized isothermal amplification methods in recent years (Yan et al., 2024 ). The Polymerase Spiral Reaction (PSR) method relies only on a single pair of primers to amplify the targeted conserved DNA sequences of various species at a constant temperature, thereby eliminating the complexities associated with other isothermal amplification methods and making them suitable for POCT. Unlike PCR, this method does not require a thermocycler, and the end products can be directly visualized by the naked eye with the aid of a suitable dye. Polymerase spiral reaction (PSR) techniques have been successfully employed to detect various meat species in adulterated meat, including pork (Danawadkar et al., 2023 ), chevon (Shree et al., 2023 ), buffalo (Jawla and Chatli, 2024 ), and cattle (Jawla et al., 2024 ) meat. However, no PSR-based technique has yet been developed to detect duck meat. The current study aimed to develop a PSR-based isothermal amplification assay that could be visualized with the naked eye using a suitable dye for the detection of duck meat in adulterated meat samples of other species when duck meat is used as an adulterant. 2. Materials and methods 2.1 Sample collection Fresh duck and meat samples of other species (chicken, quail, turkey, cattle, buffalo, sheep, goat, and pig) were collected from slaughterhouses located in the Asansol, Paschim Bardhaman, Bankura, and Birbhum districts of West Bengal, India. To prevent cross-contamination, all meat samples were collected under strict protocols using separate sterile gloves and containers for each meat species. Several processed duck meat samples were collected from various authenticated and homegrown retailers at different locations in the Paschim Bardhaman, Bankura, and Birbhum districts of West Bengal, India. Heat-treated and frozen duck meat at various temperature and time combinations was prepared in the laboratory under aseptic conditions. Each sample container was sealed properly and marked with the collection date, time, location, and species name of the sample at the time of collection. Samples were collected on different dates and transported to the laboratory in a contamination-free environment for analysis. Once the meat samples were transferred to the laboratory, they were stored at -20°C. 2.2 Reagents and kits The DNeasy Blood and Tissue Kit (QIAGEN, Germany) was used for DNA extraction. DreamTaq PCR Master Mix (ThermoFisher Scientific, USA) was used for PCR. UltraPure™ Agarose (ThermoFisher Scientific, USA) was used for gel electrophoresis. Various reagents and components were used in the PSR assay, such as 10X reaction buffer, betaine, Lyo-ready Bst DNA Polymerase, Tris-acetate-EDTA buffer, DNase-free water, GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific, USA), magnesium sulfate (MgSO 4 ) from New England Biolabs (USA), and dNTP mix from Promega. For naked-eye visualization, acridine orange (AO), hydroxynaphthol blue (HNB), SYBR Gold, and SYBR Green I dye were used. 2.3 Genomic DNA Extraction DNA was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's protocol. Extracted genomic DNA samples were stored at -20°C to preserve them for long-term preservation. The purity of the DNA samples ranged from 1.8 to 2.0 (OD 260/280), and the concentrations were 100–130 ng/µL for fresh, heat-treated, and binary mixtures of meat samples, and 90–100 ng/µL for processed meat samples. 2.4 Primers designing for PCR and PSR Duck-specific PCR and PSR primers were designed by marking a highly conserved region of the 12S rRNA gene (GenBank accession number: KF469286.1) in Anas platyrhynchos . PCR primers were designed using the NCBI Primer-BLAST tool, and specificity assessment was performed using NCBI-BLAST and IDT-OligoAnalyzer to confirm the effectiveness of the primers in targeting the desired gene sequence. PSR primers were finalized by attaching a specific sequence to the 5’ end of both the forward and reverse primers designed for PCR. All primer sets were manufactured by Eurofins Genomic India Pvt. Ltd. The complete primer sequences used in all the assays are listed in Table 1 . Table 1 Oligonucleotides used in amplification assays targeting the 12S rRNA gene. Name of the assay Primer code Sequences Product length PCR PCR-AP-12SrR-FP 5’-ACCGAAGTATCCGCCAGAGA-3’ 187 bp PCR-AP-12SrR-RP 5’-CCACTGTTGCGCTCTCATTC-3’ PSR PSR-AP-12SrR-FP 5’-acgattcgtacatagaagtatagACCGAAGTATCCGCCAGAGA-3’ Variable PSR-AP-12SrR-RP 5’-gatatgaagatacatgcttagcaCCACTGTTGCGCTCTCATTC-3’ 2.5 Optimization of PCR and PSR assay The reaction mixture for the PCR assay was prepared to a volume of 25 µL, which consisted of 12.5 µL of DreamTaq Green PCR Master Mix, 1 µL of each primer (10 pmol), and 1 µL of duck DNA template, and the remaining volume was adjusted by adding nuclease-free water (NFW) to a total volume of 25 µL. DNA amplification was performed using a Bio-Rad T100 Thermal Cycler. The PCR protocol consisted of an initial denaturation step at 95°C for 3 min, followed by 35 cycles of amplification with denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min. A final extension step was performed at 72°C for 5 min. The resulting PCR products were analyzed by electrophoresis on a 1.3% agarose gel to assess amplification and identify the presence of amplified DNA. The reaction mixture for the PSR assay was prepared in a volume of 25 µL and contained a wide range of reagents and components for precise optimization. Various parameters were analyzed, such as primer concentrations range from 1 to 100 pmol µL − 1 , dNTPs concentrations ranging from 1 to 1.6 mM, Lyo-ready Bst DNA Polymerase concentrations from 2–8 U, magnesium sulfate (MgSO₄) concentrations from 1-8mM, betaine concentrations from 0.4-1.0 M, temperature gradient of 60–70℃ and incubation time ranging from 54–70 minutes. The remaining volume was adjusted by adding nuclease-free water (NFW) to a total volume of 25 µL. 2.6 Naked-eye detection of the amplified PSR product by use of dyes For naked-eye visualization and endpoint detection, four dyes were separately tested to visualize the PSR product. AO at a 1:10 dilution of a 1 mg/mL solution was added at a rate of 1 µl before the start of the reaction. HNB was added to the PSR reaction mixtures at a concentration of 240 µM and a volume of 1 µL before the start of isothermal amplification. SYBR Gold was diluted 1:20 and added at a volume of 1 µl to the post-amplification reaction mixture. 1 µl of SYBR Green I at a dilution of 1:20 was added to the PSR amplified product reaction mixture. To further assess the effectiveness of all four dyes, amplified PSR products were evaluated by 1.3% agarose gel electrophoresis to observe a ladder-like pattern upon successful amplification. 2.7 Specificity check The specificity of both PCR and PSR assays was confirmed, with no cross-reactivity with the DNA of other meat species, such as chicken, quail, turkey, cattle, buffalo, sheep, goats, and pigs. The specificity was tested three times to verify the repeatability of the results. 2.8 Sensitivity check The analytical sensitivity of the PSR assay was determined using DNA from duck tissues. For this purpose, 30ng/µL of the DNA sample was serially diluted tenfold for ten times using 1X TE buffer.​ The sensitivity of the PCR assay was determined by using the same DNA dilution range. Both assays were used for parallel comparisons and to assess the optimal sensitivity of the PSR. A reaction mixture without template DNA was used as a negative control to confirm the absence of false positive results. Sensitivity testing was performed thrice to verify the repeatability of the detected results. 2.9 Validation of PSR assay 2.9.1 Heat-treated samples at different temperatures and times Raw duck meat samples were subjected to heat treatment, maintaining a contamination-free environment in the laboratory with different time and temperature combinations. Meat samples were heat-treated at 70, 80, 90, 100, and 121°C for 5, 10, 20, 30, 45, and 60 min. Thus, 30 meat samples were prepared, from which DNA was extracted for further study. 2.9.2 Processed, cooked, and frozen samples Four processed duck meat samples (sausage, smoked meat, minced meat, and patty) were collected from local retailers. Four raw duck meat samples were baked at 190℃ for 10, 15, 20, and 25 min; four raw duck meat samples were fried at 130–150℃ for 3, 6, 9, and 12 min; four raw duck meat samples were cooked in mustard oil with 10 min marination of salt, turmeric, ginger-garlic paste, and garam masala powder at 120–130℃ for 10, 15, 20, and 25 min; and four raw duck meat samples were stored at -20℃ for 1, 2, 4, and 6 months. After obtaining 20 such meat samples, DNA was extracted for further study. 2.9.3 Different combinations of binary meat admixture Meat admixtures of duck meat and sheep meat were formulated in different combinations at ratios of 90:10, 75:25, 50:50, 25:75, 10:90, 05:95, 01:99, 0.5:99.5, 0.1:99.9, and 0.01:99.99, each weighing one gram, to test the ability of PSR to trace adulteration to the tested extent. After proper blending, DNA was extracted for further PSR analysis. 3. Results and discussion 3.1 Optimization of the PSR assay PSR primers were used to identify the optimum concentrations in the range of 1–100 pmol µL ⁻1 . The results for 10 and 100 pmol µL ⁻ 1 showed the best and similar intensities for both dye-based naked-eye detection and agarose gel electrophoresis. Positive amplification showed visible dye color changes, such as HNB turning blue from purple under normal light (Fig. 1 .A.a.i), and AO turning yellowish-green fluorescent from orange under UV light (Fig. 1 .A.a.ii), SYBR Gold turning to golden-greenish fluorescence from colorless under UV light (Fig. 1 .A.a.iii), SYBR Green I turned green fluorescent from colorless under UV light (Fig. 1 .A.a.iv). Agarose gel electrophoresis of the amplified PSR product showed a clear ladder-like pattern in confined lanes with primer concentrations of 10 and 100 pmol µL ⁻ 1 (Fig. 1 .B.a.). However, in the case of 1 pmol µL ⁻ 1 concentration, a weak and faded ladder pattern was observed (Fig. 1 .B.a) and a minimal color change occurred (Fig. 1 .A.a.i-iv). A primer concentration of 10 pmol µL ⁻ 1 was considered sufficient to ensure precise target binding and reduce nonspecific amplification. The assessment of dNTPs concentrations in a series of 1, 1.2, 1.4, and 1.6 mM for the PSR assay was validated with visible color changes using all the dyes (Fig. 1 .A.b.i-iv). But in case of agarose gel electrophoresis for the same, prominent ladder patterns were obtained at 1 and 1.2 mM, and faded patterns were observed at 1.4 and 1.6 mM (Fig. 1 .B.b). From these observations, 1mM dNTPs concentration was found to be the best fit. In previous studies, it has been reported that dNTPs concentration is affected by Mg 2+ concentration, oligonucleotide length, and incubation temperature in the PSR assay (Roux, 2009 ). In some studies, higher concentrations have also been utilized by previous researchers for the same assay (Momin et al., 2020 ; Liu et al., 2023 ). Lyo-ready Bst DNA Polymerase concentrations of 2, 4, 6, and 8 U were tested for the PSR assay with the formation of a distinct color shift in all the dyes (Fig. 1 .A.c.i-iv); however, in agarose gel electrophoresis, prominent ladder patterns were obtained at 2 and 4 U, and gradually faded patterns were observed at 6 and 8 U (Fig. 1 .B.c). Depending on the results, 2 U lyo-ready Bst DNA Polymerase was marked as the optimal concentration for duck-specific 12S rRNA gene PSR amplification. Magnesium sulfate (MgSO₄) concentrations of 1, 2, 4, 6, and 8 mM were determined using the PSR assay. A color shift with lower intensity was observed for all dyes (Fig. 1 .A.d.i-iv), and a weak ladder pattern on agarose gel electrophoresis (Fig. 1 .B.d) were observed at 2 and 4 mM concentrations, and no color change was observed in any of the dyes (Fig. 1 .A.d.i-iv), or no ladder pattern was observed (Fig. 1 .B.d) at a concentration of 1 mM. However, a stronger change in the color intensity of all the dyes was observed (Fig. 1 .A.d.i-iv), and distinct ladder patterns on agarose gel electrophoresis (Fig. 1 .B.d) were observed at 6 and 8 mM concentrations. Therefore, a 6 mM concentration of MgSO₄ was chosen for a successful PSR assay. MgSO₄ plays a crucial role in enhancing the amplification efficacy, as it has a contradictory relation with Bst DNA polymerase. Betaine concentrations of 0.4, 0.6, 0.8, and 1.0 M were used to standardize the PSR assay. A stronger color change was observed for all dyes (Fig. 1 .A.e.i-iv), and a distinct ladder pattern on agarose gel electrophoresis (Fig. 1 .B.e) were observed in 0.8 and 1.0 M, while faded color changes were observed for all dyes (Fig. 1 .A.e.i-iv) and a weak ladder pattern on agarose gel electrophoresis (Fig. 1 .B.e) were observed in 0.4 and 0.6 M. Analysing these observations, 0.8 M concentration of betaine was found to be suitable for this PSR amplification. Betaine plays a crucial role as a zwitterionic molecule in balancing reactions to intensify species specificity (Foo et al., 2020 ). An inadequate betaine concentration may lead to false positives, as it is unable to inhibit secondary structures, while an unrestricted betaine concentration hinders the successful amplification process (Foo et al., 2020 ). Another study on microbial DNA identification (Milton et al., 2021 ) also showed that 0.8 M betaine effectively minimizes nonspecific amplification without affecting reaction kinetics. A previous study also reported lower concentrations of 0.5 M for identifying chevon (Shree et al., 2023 ). Because temperature is a crucial factor in PSR amplification, a temperature range of 60–70°C was analyzed for optimization. In this assessment, no color change was observed for any of the dyes (Fig. 1 .A. f. i-iv), and no ladder pattern on agarose gel electrophoresis (Fig. 1 .B. f.) were observed at 60°C. But at 61–63℃, gradual fading of the color change (Fig. 1 .A. f. i-iv), and a weaker ladder pattern on agarose gel electrophoresis (Fig. 1 .B. f.), were also observed. At a temperature in the 64–70℃ range, a gradual increase in the intensity of the color change for all dyes was observed (Fig. 1 .A.f.i-iv) and visible ladder patterns on agarose gel electrophoresis were observed (Fig. 1 .B.f). Therefore, 65°C was determined as the optimal temperature for the PSR assay. The PSR assay was also analyzed over a wide time range of 54–70 min. No amplification was found in 54 and 56 minutes for both dye-based (Fig. 1 .A.g.i-iv) and agarose electrophoresis methods (Fig. 1 .B.g), but faded results were observed after 58 min (Fig. 1 .A.g.i-iv and Fig. 1 .B.g). The stronger intensity of the color shift of the dyes (Fig. 1 .A.g.i-iv), and visible ladder patterns on gel electrophoresis (Fig. 1 .B.g) was consistent between 60 and 70 min. Based on these observations, 60 min was selected as the optimal reaction time for the PSR assay. Thus, for this PSR assay protocol study, a temperature of 65°C in association with an incubation time of 60 min was selected as a standardized combination to obtain successful amplification without any hindrance. 3.2 Specificity check The novel species-specific 12S rRNA primers for both PCR and PSR assays for ducks demonstrated accurate specificity with positive results in confined lanes of duck-specific samples. In the PSR assay, various dyes were applied for naked-eye detection (Fig. 2 .A.i-iv), and agarose gel electrophoresis (Fig. 2 .B.i) showed that reactions consisting of DNA samples of chicken, quail, turkey, cattle, buffalo, sheep, goat, pig, and negative control were not amplified. The repeatability of the developed PSR assay was established by repeating the test three times. These observations correspond to the PCR assay results performed with species-specific 12S rRNA primers for ducks (Fig. 2 .B.ii) and ensured high specificity for duck DNA detection. The high specificity and repeatability of the PSR assay have been reported for various target species, such as pork (Danawadkar et al., 2023 ), chevon (Shree et al., 2023 ), buffalo (Jawla and Chatli, 2024 ), and cattle (Jawla et al., 2024 ). The use of an easier primer design method and the utilization of only a pair of primers at low concentrations enable higher specificity in PSR (Liu et al., 2024 ) by reducing nonspecific amplification through accurate hybridization with the target DNA compared to the LAMP assay (Li and Fan, 2017 ). 3.3 Sensitivity check To validate the limit of detection (LOD), the sensitivity of the PSR assay was analyzed by naked-eye visualization through dye-based detection (Fig. 3 .A.i-iv), and agarose gel electrophoresis (Fig. 3 .B.i). In this naked-eye visualization analysis, a gradual decrease in the dye efficiency occurred with tenfold serial dilution of DNA concentrations (from 30 ng) for ten times, with non-traceability of the PSR end-point product below 3 femtograms (fg) concentration. With this LOD, the PSR assay showed a much higher sensitivity of up to 3 fg. Thus, the PSR assay efficiency to detect the DNA was three times that of tenfold further serial dilution in comparison to the PCR assay efficiency, which showed a sensitivity of only 3 picogram (pg) (Fig. 3 .B.ii). The repeatability of the developed PSR assay was established by repeating the test three times. The PSR assay, with a LOD of 3 fg, outperforms the sensitivity of various reported nucleic acid-based amplification methods in terms of the efficient detection of duck meat. While in comparison to the PSR assay, the multiplex PCR assay has an LOD of 0.01 ng (Yang et al., 2022 ), LAMP has LODs of 10 pg (Cho et al., 2014 ), real-time RPA has shown an LOD of 10 ng DNA, and the RPA lateral flow strip assay has an LOD of 1 pg (Zhou et al., 2023 ). 3.4 Authentication/Validation of PSR assay 3.4.1 Heat-treated samples at different temperatures and times The developed PSR assay was employed to detect duck content in heat-treated or boiled duck meat products by extracting DNA from duck meat, as described in section 2.9.1. A total of 30 samples at different time and temperature variations were used to validate the efficacy of the PSR assay over a wide range using naked-eye detection (Fig. 4 .A.i-iv), and agarose gel electrophoresis (Fig. 4 .B). All 30 samples showed successful detection of duck DNA over this range, with no amplification observed in the negative control. The application of heat while preparing processed meat may enhance the disruption of the cell membrane and aid in DNA extraction and subsequent amplification efficacy (Arun et al., 2014 ), making DNA detection-based methods more reliable for identifying adulteration in processed food. In the current study, successful amplification of heat-treated samples at temperatures of up to 121℃ for 60 min was observed. This indicates that the PSR assay is efficacious even after high processing temperatures, making it a suitable method for identifying duck content in heat-treated or processed commercial meat products. 3.4.2 Processed, cooked, and frozen samples The PSR assay was also used to detect duck content in processed, cooked, and frozen duck meat products. DNA was extracted from duck meat, as described in Section 2.9.2. The sample size was kept at 20 for different variations of cooking or freezing procedures, varying with time and temperature combinations. To validate the efficiency of the PSR assay over a wide range, we used dye-based visual detection (Fig. 5 .A.i-iv), and agarose gel electrophoresis methods (Fig. 5 .B) were performed. All 20 samples showed successful detection of duck DNA over this range, with no amplification observed in the negative control. PCR detection can be hindered by the addition of spices, salt, high-heat processing, and cooking oil, which may lead to potential inaccuracies (Yang et al., 2022 ). However, when the PSR assay was tested on such samples, it was found to be reliable and sensitive for detecting duck meat, making it more suitable for on-site detection. 3.4.3 Different combinations of binary meat admixture Duck meat was mixed with sheep meat in a series of combinations to prepare binary meat admixture as described in section 2.9.3, to validate the highly sensitive and species-specific detection nature of the PSR assay. For this, standardized naked-eye detection (Fig. 6 .A.i-iv), and agarose gel electrophoresis (Fig. 6 .B) were applied to confirm the suitability of the PSR assay with a minimum LOD of 0.01%. In previous studies, multiplex PCR reported a 1% LOD (Hou et al., 2015 ), fluorescence-based RPA at a 1% LOD (Chen et al., 2022 ), and PCR + FITC/biotin-labeled- primers with a lateral flow strip (LFS) at a 0.05% LOD (Qin et al., 2019 ) identified the duck content in the designed experimental adulteration ratio. However, this PSR assay study has yielded positive testing results, even with a 0.01% LOD, which surpasses all other methods in detecting adulteration to this extent. 4. Conclusion For the first time, a 12S rRNA gene PSR-based assay was designed for the rapid, accurate, and sensitive detection of duck meat in adulterated meat samples of other species where duck tissue has been added as an adulterant. The developed PSR assay demonstrated the capability of detecting as low as 3 fg/µL of duck genomic DNA within 60 min at 65°C. The PSR assay exhibited high specificity, with no cross-reactivity with the eight other meat species tested. The assay can identify duck meat adulteration at levels as low as 0.01% in sheep meat mixtures. The PSR assay successfully detected duck DNA in 30 combinations of heat-treated samples (up to a maximum of 121°C for 60 min) and 20 processed or frozen samples, demonstrating its robustness across various sample types. Naked-eye detection using different dyes, such as HNB, Acridine orange, SYBR Gold, and SYBR Green I, enables direct visualisation of the results. Overall, the most precise result was obtained by SYBR Green I. However, for HNB dye detection, UV light is not required, making it more suitable for point-of-care testing. The PSR method surpasses PCR and other techniques in terms of sensitivity, rapidity, and simplicity for on-site detection. The developed PSR assay exhibits high sensitivity, specificity, and suitability for point-of-care testing, making it potentially valuable for regulatory agencies and food producers to rapidly screen for meat adulteration when duck meat is used as an adulterant. Declarations Declaration of interests The authors declare that they do not have any competing financial interests or personal relationships that could have influenced the work reported in this paper. Author Contribution R.C.: Writing – original draft, writing – review and editing, investigation and formal analysis, visualization and validation. S.S.: Writing – review and editing, conceptualization, methodology, visualization and validation, project administration, supervision, and funding acquisition. Acknowledgement The authors are grateful to SERB (ANRF sanction order No. SUR/2022/002154) for funding the research on the use of PSR-based isothermal amplification for detecting meat adulteration. 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Supplementary Files Graphicalabstract.tiff Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Feb, 2026 Reviews received at journal 08 Feb, 2026 Reviewers agreed at journal 30 Jan, 2026 Reviewers invited by journal 29 Jan, 2026 Editor assigned by journal 21 Jan, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 17 Jan, 2026 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. 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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-8627709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":582916877,"identity":"11fd20b5-3876-4ced-8255-a12d0674fc56","order_by":0,"name":"Ranjita Chatterjee","email":"","orcid":"","institution":"Kazi Nazrul University","correspondingAuthor":false,"prefix":"","firstName":"Ranjita","middleName":"","lastName":"Chatterjee","suffix":""},{"id":582916878,"identity":"c2a2a8b9-ee76-46a3-b853-79eb7e20f44b","order_by":1,"name":"Sourabh Sulabh","email":"data:image/png;base64,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","orcid":"","institution":"Kazi Nazrul University","correspondingAuthor":true,"prefix":"","firstName":"Sourabh","middleName":"","lastName":"Sulabh","suffix":""}],"badges":[],"createdAt":"2026-01-17 18:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8627709/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8627709/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101656137,"identity":"7177ad60-3ec6-4477-959d-7143bd924893","added_by":"auto","created_at":"2026-02-02 10:07:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of polymerase spiral reaction (PSR) assay.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Dye-based detection for optimization of PSR assay with various parameters of concentration of \u003cstrong\u003e(a)\u003c/strong\u003e Primer,\u003cstrong\u003e (b) \u003c/strong\u003edNTPs,\u003cstrong\u003e (c)\u003c/strong\u003e Bst DNA polymerase, \u003cstrong\u003e(d) \u003c/strong\u003eMgSO₄, \u003cstrong\u003e(e) \u003c/strong\u003eBetaine, \u003cstrong\u003e(f) \u003c/strong\u003eTemperature, and \u003cstrong\u003e(g)\u003c/strong\u003e Time with different dyes: \u003cstrong\u003ei.\u003c/strong\u003e HNB (under white light); \u003cstrong\u003eii.\u003c/strong\u003e Acridine orange, \u003cstrong\u003eiii.\u003c/strong\u003e SYBR Gold, and \u003cstrong\u003eiv.\u003c/strong\u003eSYBR Green I (under UV light) with optimized values is marked with a dotted box. \u003cstrong\u003eB.\u003c/strong\u003e Agarose gel electrophoresis for optimization of PSR assay with various parameters of \u003cstrong\u003ea-g\u003c/strong\u003ewith 100 bp ladder \u003cstrong\u003e(L)\u003c/strong\u003e and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/11efb395d9272dcb7dddc6eb.jpg"},{"id":101656142,"identity":"069af010-15a2-40cb-b161-7ee00248f8fb","added_by":"auto","created_at":"2026-02-02 10:07:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of the specificity of the Polymerase Spiral Reaction (PSR) assay.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eDye based detection for validation of specificity in meat different of animals such as \u003cstrong\u003e(D) \u003c/strong\u003eDuck, \u003cstrong\u003e(T) \u003c/strong\u003eTurkey, \u003cstrong\u003e(Q)\u003c/strong\u003e Quail, \u003cstrong\u003e(Ch) \u003c/strong\u003eChicken, \u003cstrong\u003e(C) \u003c/strong\u003eCattle, \u003cstrong\u003e(B) \u003c/strong\u003eBuffalo, \u003cstrong\u003e(P) \u003c/strong\u003ePig, \u003cstrong\u003e(S) \u003c/strong\u003eSheep, \u003cstrong\u003e(G) \u003c/strong\u003eGoat and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e with different dyes: \u003cstrong\u003ei.\u003c/strong\u003e HNB (under white light); \u003cstrong\u003eii.\u003c/strong\u003e Acridine orange, \u003cstrong\u003eiii.\u003c/strong\u003e SYBR Gold, and \u003cstrong\u003eiv.\u003c/strong\u003eSYBR Green I (under UV light) with specified species marked in a dotted box. \u003cstrong\u003eB.\u003c/strong\u003eAgarose gel electrophoresis for validation of specificity in meat of different animals \u003cstrong\u003e(D, T, Q, Ch, C, B, P, S, \u003c/strong\u003eand \u003cstrong\u003eG) \u003c/strong\u003ein both \u003cstrong\u003ei. \u003c/strong\u003ePSR and \u003cstrong\u003eii.\u003c/strong\u003e PCR assay with 100 bp ladder \u003cstrong\u003e(L)\u003c/strong\u003e and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/deaa0cf0771b9e3df73ea26f.jpg"},{"id":101656143,"identity":"558a90c1-2942-4ec1-8583-627caad24b42","added_by":"auto","created_at":"2026-02-02 10:07:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of the sensitivity of the Polymerase Spiral Reaction (PSR) assay.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Dye-based detection for validation of the sensitivity of duck DNA samples at decreasing concentrations from \u003cstrong\u003e30ng-30ag\u003c/strong\u003ewith different dyes: \u003cstrong\u003ei.\u003c/strong\u003e HNB (under white light); \u003cstrong\u003eii.\u003c/strong\u003e Acridine orange, \u003cstrong\u003eiii.\u003c/strong\u003e SYBR Gold, and \u003cstrong\u003eiv.\u003c/strong\u003eSYBR Green I (under UV light). \u003cstrong\u003eB.\u003c/strong\u003e Agarose gel electrophoresis for validation of the sensitivity of duck DNA samples in both \u003cstrong\u003ei.\u003c/strong\u003e PSR assay at decreasing concentrations from \u003cstrong\u003e30ng-30ag\u003c/strong\u003e and \u003cstrong\u003eii.\u003c/strong\u003e PCR assay at decreasing concentrations from \u003cstrong\u003e30ng-30ag\u003c/strong\u003e with 100 bp ladder \u003cstrong\u003e(L)\u003c/strong\u003eand Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/24aa28f880688704b2681b79.jpg"},{"id":101656140,"identity":"03d72a21-39a9-4e76-b44c-373bceaa09ff","added_by":"auto","created_at":"2026-02-02 10:07:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of Polymerase Spiral Reaction (PSR) assay for detecting various heat-treated samples. A.\u003c/strong\u003eDye based detection for validation of various heat-treated samples in different temperature and time combination of \u003cstrong\u003e(1-6) \u003c/strong\u003eheat-treated meat samples at 70℃ for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes respectively, \u003cstrong\u003e(7-12) \u003c/strong\u003eheat-treated meat samples at 80℃ for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes respectively, \u003cstrong\u003e(13-18\u003c/strong\u003e) heat-treated meat samples at 90℃ for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes respectively, \u003cstrong\u003e(19-24) \u003c/strong\u003eheat-treated meat samples at 100℃ for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes respectively, \u003cstrong\u003e(25-30) \u003c/strong\u003eheat-treated meat samples at 121℃ for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes respectively and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003ewith different dyes: \u003cstrong\u003ei.\u003c/strong\u003e HNB (under white light); \u003cstrong\u003eii.\u003c/strong\u003e Acridine orange, \u003cstrong\u003eiii.\u003c/strong\u003e SYBR Gold, and \u003cstrong\u003eiv.\u003c/strong\u003eSYBR Green I (under UV light). \u003cstrong\u003eB.\u003c/strong\u003e Agarose gel electrophoresis for validation of various heat-treated samples in different temperature and time combinations of \u003cstrong\u003e1-30\u003c/strong\u003ewith 100 bp ladder \u003cstrong\u003e(L)\u003c/strong\u003e and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/ab99ae771f209a888762fc16.jpg"},{"id":101656138,"identity":"b17e2f5e-7192-4c6a-afd2-73612dc92d10","added_by":"auto","created_at":"2026-02-02 10:07:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of Polymerase Spiral Reaction (PSR) assay for detecting various processed, cooked, and frozen samples. A.\u003c/strong\u003e Dye based detection for validation of various processed, cooked and frozen samples in different variety of \u003cstrong\u003e(1-4) \u003c/strong\u003eprocessed meat products from market, \u003cstrong\u003e(5-8) \u003c/strong\u003emeat samples baked at 190℃ for 10 minutes, 15 minutes, 20 minutes and 25 minutes respectively, \u003cstrong\u003e(9-12\u003c/strong\u003e) meat samples fried at 130-150℃ for 3 minutes, 6 minutes, 9 minutes and 12 minutes respectively, \u003cstrong\u003e(13-16) \u003c/strong\u003emeat samples cooked with Indian spices at 120-130℃ for 10minutes, 15 minutes, 20 minutes and 25 minutes respectively, \u003cstrong\u003e(17-20) \u003c/strong\u003emeat samples stored at -20℃ for 1 month, 2 months, 4 months and 6 months respectively and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e with different dyes: \u003cstrong\u003ei.\u003c/strong\u003eHNB (under white light); \u003cstrong\u003eii.\u003c/strong\u003e Acridine orange, \u003cstrong\u003eiii.\u003c/strong\u003e SYBR Gold, and \u003cstrong\u003eiv.\u003c/strong\u003e SYBR Green I (under UV light). \u003cstrong\u003eB.\u003c/strong\u003e Agarose gel electrophoresis for validation of various processed, cooked, and frozen samples in different varieties of \u003cstrong\u003e1-20\u003c/strong\u003ewith 100 bp ladder \u003cstrong\u003e(L)\u003c/strong\u003e and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/a34ac6fd667e957d89f4b8c5.jpg"},{"id":101656141,"identity":"4b2eb948-fc12-4e8f-8204-1fbb50c44f35","added_by":"auto","created_at":"2026-02-02 10:07:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of Polymerase Spiral Reaction (PSR) assay for detecting binary meat admixtures.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eDye based detection for validation of binary meat admixtures in different combinations in ratios of \u003cstrong\u003e(1) \u003c/strong\u003e90:10, \u003cstrong\u003e(2) \u003c/strong\u003e75:25, \u003cstrong\u003e(3) \u003c/strong\u003e50:50, \u003cstrong\u003e(4) \u003c/strong\u003e25:75, \u003cstrong\u003e(5) \u003c/strong\u003e10:90, \u003cstrong\u003e(6) \u003c/strong\u003e05:95, \u003cstrong\u003e(7) \u003c/strong\u003e01:99, \u003cstrong\u003e(8) \u003c/strong\u003e0.5:99.5, \u003cstrong\u003e(9) \u003c/strong\u003e0.1:99.9, \u003cstrong\u003e(10) \u003c/strong\u003e0.01:99.99 and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e with different dyes: \u003cstrong\u003ei.\u003c/strong\u003e HNB (under white light); \u003cstrong\u003eii.\u003c/strong\u003e Acridine orange, \u003cstrong\u003eiii.\u003c/strong\u003e SYBR Gold, and \u003cstrong\u003eiv.\u003c/strong\u003e SYBR Green I (under UV light). \u003cstrong\u003eB.\u003c/strong\u003eAgarose gel electrophoresis for validation of binary meat admixtures in different combinations in ratios of \u003cstrong\u003e1-10\u003c/strong\u003ewith 100 bp ladder \u003cstrong\u003e(L)\u003c/strong\u003e and Negative Control \u003cstrong\u003e(NC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/2ebee162a4008f49b181a04f.jpg"},{"id":101753229,"identity":"787684e6-e3f4-4533-bb81-063af81a8a31","added_by":"auto","created_at":"2026-02-03 10:39:29","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":319157,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8627709/v1/b1aa25d2db5947a39500ffcf.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Food Authenticity: A Dye-Based Polymerase Spiral Reaction for Rapid Detection of Duck Tissue as Adulterants in Meat Samples","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMeat and meat products play an important role in a balanced meat-based diet and significantly contribute to the necessary protein intake in the daily diet. Meat adulteration has become an important issue as it affects food safety, consumer trust, and market regulation worldwide (Wang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite all food safety laws and protocols, fraud in meat and meat products has the highest number of cases among all types (Marvin et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to market surveys in Greece, the U.S, Canada, and China, this malpractice is a crucial global scenario (Stamatis et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kane and Hellberg, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shehata et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This type of adulteration in meat and meat products may not only cause economic loss to consumers but also lead to severe health problems, raising health concerns.\u003c/p\u003e \u003cp\u003eDuck meat is frequently mixed as an adulterant in other commercial meats, such as beef, lamb, or chicken, because of its low cost and similar texture and appearance (Zhou et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In China, butter, mutton, or beef essence is used to cover up the duck odor, especially in processed items such as skewers or sausages, so that they can be easily substituted for authentic meat or meat products (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The Mislabelling of meat products when the most common adulterant was duck meat has also been reported (Song et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This is a clear case of economic fraud as consumers pay higher prices for cheaper meat. In studies, it has been found that individuals with avian meat allergies, such as \u0026ldquo;bird\u0026ndash;egg syndrome, and persons reacting to one bird species' meat (e.g., chicken) can also react to duck meat, probably due to the presence of similar cross-reacting proteins having binding capabilities with IgE antibodies (Kelso et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). This reaction can lead to skin rashes, itching, respiratory problems, or even anaphylaxis in severe cases (Wanniang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Histamine accumulation in the consumer's body causes the main clinical symptoms such as itching, swelling, headaches, gastrointestinal issues, and bronchospasm (Lisiecka, M. Z., 2025).\u003c/p\u003e \u003cp\u003eSeveral methods, such as chromatography (Di Stefano et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and spectroscopy (Weng et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), have been used to detect meat adulteration; however, their use in the food industry is very limited. However, several protein- and DNA-based methods (He et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) have emerged as crucial techniques for rapid and accurate detection. Enzyme-linked immunosorbent assay (ELISA) is a less time-consuming and easy-to-perform method; however, its ability to distinguish between closely related species is unsatisfactory, and it is less effective in checking the authenticity of processed meat products as the target protein denatures during harsh processing conditions. Polymerase Chain Reaction (PCR) is a DNA-based method that detects meat adulteration more rapidly and with better accuracy; in particular, real-time PCR is more efficient for detecting processed meat products as DNA is more thermostable. However, the entire PCR procedure is performed under precise temperature control with specialized equipment, making it inconvenient to perform without a proper laboratory setup and not a great choice for point-of-care testing (POCT) (Perestam et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNucleic acid-based isothermal amplification techniques are categorized based on various reaction principles and are used for the detection of meat adulteration. According to previous studies, LAMP, SEA, CPA, and RPA have been the four most frequently utilized isothermal amplification methods in recent years (Yan et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Polymerase Spiral Reaction (PSR) method relies only on a single pair of primers to amplify the targeted conserved DNA sequences of various species at a constant temperature, thereby eliminating the complexities associated with other isothermal amplification methods and making them suitable for POCT. Unlike PCR, this method does not require a thermocycler, and the end products can be directly visualized by the naked eye with the aid of a suitable dye.\u003c/p\u003e \u003cp\u003ePolymerase spiral reaction (PSR) techniques have been successfully employed to detect various meat species in adulterated meat, including pork (Danawadkar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), chevon (Shree et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), buffalo (Jawla and Chatli, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and cattle (Jawla et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) meat. However, no PSR-based technique has yet been developed to detect duck meat. The current study aimed to develop a PSR-based isothermal amplification assay that could be visualized with the naked eye using a suitable dye for the detection of duck meat in adulterated meat samples of other species when duck meat is used as an adulterant.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection\u003c/h2\u003e \u003cp\u003eFresh duck and meat samples of other species (chicken, quail, turkey, cattle, buffalo, sheep, goat, and pig) were collected from slaughterhouses located in the Asansol, Paschim Bardhaman, Bankura, and Birbhum districts of West Bengal, India. To prevent cross-contamination, all meat samples were collected under strict protocols using separate sterile gloves and containers for each meat species. Several processed duck meat samples were collected from various authenticated and homegrown retailers at different locations in the Paschim Bardhaman, Bankura, and Birbhum districts of West Bengal, India. Heat-treated and frozen duck meat at various temperature and time combinations was prepared in the laboratory under aseptic conditions. Each sample container was sealed properly and marked with the collection date, time, location, and species name of the sample at the time of collection. Samples were collected on different dates and transported to the laboratory in a contamination-free environment for analysis. Once the meat samples were transferred to the laboratory, they were stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Reagents and kits\u003c/h2\u003e \u003cp\u003eThe DNeasy Blood and Tissue Kit (QIAGEN, Germany) was used for DNA extraction. DreamTaq PCR Master Mix (ThermoFisher Scientific, USA) was used for PCR. UltraPure\u0026trade; Agarose (ThermoFisher Scientific, USA) was used for gel electrophoresis. Various reagents and components were used in the PSR assay, such as 10X reaction buffer, betaine, Lyo-ready Bst DNA Polymerase, Tris-acetate-EDTA buffer, DNase-free water, GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific, USA), magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e) from New England Biolabs (USA), and dNTP mix from Promega. For naked-eye visualization, acridine orange (AO), hydroxynaphthol blue (HNB), SYBR Gold, and SYBR Green I dye were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Genomic DNA Extraction\u003c/h2\u003e \u003cp\u003eDNA was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's protocol. Extracted genomic DNA samples were stored at -20\u0026deg;C to preserve them for long-term preservation. The purity of the DNA samples ranged from 1.8 to 2.0 (OD 260/280), and the concentrations were 100\u0026ndash;130 ng/\u0026micro;L for fresh, heat-treated, and binary mixtures of meat samples, and 90\u0026ndash;100 ng/\u0026micro;L for processed meat samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Primers designing for PCR and PSR\u003c/h2\u003e \u003cp\u003eDuck-specific PCR and PSR primers were designed by marking a highly conserved region of the 12S rRNA gene (GenBank accession number: KF469286.1) in \u003cem\u003eAnas platyrhynchos\u003c/em\u003e. PCR primers were designed using the NCBI Primer-BLAST tool, and specificity assessment was performed using NCBI-BLAST and IDT-OligoAnalyzer to confirm the effectiveness of the primers in targeting the desired gene sequence. PSR primers were finalized by attaching a specific sequence to the 5\u0026rsquo; end of both the forward and reverse primers designed for PCR. All primer sets were manufactured by Eurofins Genomic India Pvt. Ltd. The complete primer sequences used in all the assays are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eOligonucleotides used in amplification assays targeting the 12S rRNA gene.\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\u003eName of the assay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProduct length\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ePCR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCR-AP-12SrR-FP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ACCGAAGTATCCGCCAGAGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e187 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCR-AP-12SrR-RP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCACTGTTGCGCTCTCATTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ePSR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePSR-AP-12SrR-FP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-acgattcgtacatagaagtatagACCGAAGTATCCGCCAGAGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePSR-AP-12SrR-RP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-gatatgaagatacatgcttagcaCCACTGTTGCGCTCTCATTC-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 \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Optimization of PCR and PSR assay\u003c/h2\u003e \u003cp\u003eThe reaction mixture for the PCR assay was prepared to a volume of 25 \u0026micro;L, which consisted of 12.5 \u0026micro;L of DreamTaq Green PCR Master Mix, 1 \u0026micro;L of each primer (10 pmol), and 1 \u0026micro;L of duck DNA template, and the remaining volume was adjusted by adding nuclease-free water (NFW) to a total volume of 25 \u0026micro;L. DNA amplification was performed using a Bio-Rad T100 Thermal Cycler. The PCR protocol consisted of an initial denaturation step at 95\u0026deg;C for 3 min, followed by 35 cycles of amplification with denaturation at 95\u0026deg;C for 30 s, annealing at 58\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 1 min. A final extension step was performed at 72\u0026deg;C for 5 min. The resulting PCR products were analyzed by electrophoresis on a 1.3% agarose gel to assess amplification and identify the presence of amplified DNA.\u003c/p\u003e \u003cp\u003eThe reaction mixture for the PSR assay was prepared in a volume of 25 \u0026micro;L and contained a wide range of reagents and components for precise optimization. Various parameters were analyzed, such as primer concentrations range from 1 to 100 pmol \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, dNTPs concentrations ranging from 1 to 1.6 mM, Lyo-ready Bst DNA Polymerase concentrations from 2\u0026ndash;8 U, magnesium sulfate (MgSO₄) concentrations from 1-8mM, betaine concentrations from 0.4-1.0 M, temperature gradient of 60\u0026ndash;70℃ and incubation time ranging from 54\u0026ndash;70 minutes. The remaining volume was adjusted by adding nuclease-free water (NFW) to a total volume of 25 \u0026micro;L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Naked-eye detection of the amplified PSR product by use of dyes\u003c/h2\u003e \u003cp\u003eFor naked-eye visualization and endpoint detection, four dyes were separately tested to visualize the PSR product.\u003c/p\u003e \u003cp\u003e \u003col style=\"list-style-type:lower-alpha;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAO at a 1:10 dilution of a 1 mg/mL solution was added at a rate of 1 \u0026micro;l before the start of the reaction.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eHNB was added to the PSR reaction mixtures at a concentration of 240 \u0026micro;M and a volume of 1 \u0026micro;L before the start of isothermal amplification.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSYBR Gold was diluted 1:20 and added at a volume of 1 \u0026micro;l to the post-amplification reaction mixture.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e1 \u0026micro;l of SYBR Green I at a dilution of 1:20 was added to the PSR amplified product reaction mixture.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eTo further assess the effectiveness of all four dyes, amplified PSR products were evaluated by 1.3% agarose gel electrophoresis to observe a ladder-like pattern upon successful amplification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Specificity check\u003c/h2\u003e \u003cp\u003eThe specificity of both PCR and PSR assays was confirmed, with no cross-reactivity with the DNA of other meat species, such as chicken, quail, turkey, cattle, buffalo, sheep, goats, and pigs. The specificity was tested three times to verify the repeatability of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Sensitivity check\u003c/h2\u003e \u003cp\u003eThe analytical sensitivity of the PSR assay was determined using DNA from duck tissues. For this purpose, 30ng/\u0026micro;L of the DNA sample was serially diluted tenfold for ten times using 1X TE buffer.​ The sensitivity of the PCR assay was determined by using the same DNA dilution range. Both assays were used for parallel comparisons and to assess the optimal sensitivity of the PSR. A reaction mixture without template DNA was used as a negative control to confirm the absence of false positive results. Sensitivity testing was performed thrice to verify the repeatability of the detected results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Validation of PSR assay\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 Heat-treated samples at different temperatures and times\u003c/h2\u003e \u003cp\u003eRaw duck meat samples were subjected to heat treatment, maintaining a contamination-free environment in the laboratory with different time and temperature combinations. Meat samples were heat-treated at 70, 80, 90, 100, and 121\u0026deg;C for 5, 10, 20, 30, 45, and 60 min. Thus, 30 meat samples were prepared, from which DNA was extracted for further study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2 Processed, cooked, and frozen samples\u003c/h2\u003e \u003cp\u003eFour processed duck meat samples (sausage, smoked meat, minced meat, and patty) were collected from local retailers. Four raw duck meat samples were baked at 190℃ for 10, 15, 20, and 25 min; four raw duck meat samples were fried at 130\u0026ndash;150℃ for 3, 6, 9, and 12 min; four raw duck meat samples were cooked in mustard oil with 10 min marination of salt, turmeric, ginger-garlic paste, and garam masala powder at 120\u0026ndash;130℃ for 10, 15, 20, and 25 min; and four raw duck meat samples were stored at -20℃ for 1, 2, 4, and 6 months. After obtaining 20 such meat samples, DNA was extracted for further study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3 Different combinations of binary meat admixture\u003c/h2\u003e \u003cp\u003eMeat admixtures of duck meat and sheep meat were formulated in different combinations at ratios of 90:10, 75:25, 50:50, 25:75, 10:90, 05:95, 01:99, 0.5:99.5, 0.1:99.9, and 0.01:99.99, each weighing one gram, to test the ability of PSR to trace adulteration to the tested extent. After proper blending, DNA was extracted for further PSR analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Optimization of the PSR assay\u003c/h2\u003e \u003cp\u003ePSR primers were used to identify the optimum concentrations in the range of 1\u0026ndash;100 pmol \u0026micro;L\u003csup\u003e⁻1\u003c/sup\u003e. The results for 10 and 100 pmol \u0026micro;L\u003csup\u003e⁻ 1\u003c/sup\u003e showed the best and similar intensities for both dye-based naked-eye detection and agarose gel electrophoresis. Positive amplification showed visible dye color changes, such as HNB turning blue from purple under normal light (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.a.i), and AO turning yellowish-green fluorescent from orange under UV light (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.a.ii), SYBR Gold turning to golden-greenish fluorescence from colorless under UV light (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.a.iii), SYBR Green I turned green fluorescent from colorless under UV light (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.a.iv). Agarose gel electrophoresis of the amplified PSR product showed a clear ladder-like pattern in confined lanes with primer concentrations of 10 and 100 pmol \u0026micro;L\u003csup\u003e⁻ 1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.a.). However, in the case of 1 pmol \u0026micro;L\u003csup\u003e⁻ 1\u003c/sup\u003e concentration, a weak and faded ladder pattern was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.a) and a minimal color change occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.a.i-iv). A primer concentration of 10 pmol \u0026micro;L ⁻ 1 was considered sufficient to ensure precise target binding and reduce nonspecific amplification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe assessment of dNTPs concentrations in a series of 1, 1.2, 1.4, and 1.6 mM for the PSR assay was validated with visible color changes using all the dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.b.i-iv). But in case of agarose gel electrophoresis for the same, prominent ladder patterns were obtained at 1 and 1.2 mM, and faded patterns were observed at 1.4 and 1.6 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.b). From these observations, 1mM dNTPs concentration was found to be the best fit. In previous studies, it has been reported that dNTPs concentration is affected by Mg\u003csup\u003e2+\u003c/sup\u003e concentration, oligonucleotide length, and incubation temperature in the PSR assay (Roux, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In some studies, higher concentrations have also been utilized by previous researchers for the same assay (Momin et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLyo-ready Bst DNA Polymerase concentrations of 2, 4, 6, and 8 U were tested for the PSR assay with the formation of a distinct color shift in all the dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.c.i-iv); however, in agarose gel electrophoresis, prominent ladder patterns were obtained at 2 and 4 U, and gradually faded patterns were observed at 6 and 8 U (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.c). Depending on the results, 2 U lyo-ready Bst DNA Polymerase was marked as the optimal concentration for duck-specific 12S rRNA gene PSR amplification.\u003c/p\u003e \u003cp\u003eMagnesium sulfate (MgSO₄) concentrations of 1, 2, 4, 6, and 8 mM were determined using the PSR assay. A color shift with lower intensity was observed for all dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.d.i-iv), and a weak ladder pattern on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.d) were observed at 2 and 4 mM concentrations, and no color change was observed in any of the dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.d.i-iv), or no ladder pattern was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.d) at a concentration of 1 mM. However, a stronger change in the color intensity of all the dyes was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.d.i-iv), and distinct ladder patterns on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.d) were observed at 6 and 8 mM concentrations. Therefore, a 6 mM concentration of MgSO₄ was chosen for a successful PSR assay. MgSO₄ plays a crucial role in enhancing the amplification efficacy, as it has a contradictory relation with Bst DNA polymerase.\u003c/p\u003e \u003cp\u003eBetaine concentrations of 0.4, 0.6, 0.8, and 1.0 M were used to standardize the PSR assay. A stronger color change was observed for all dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.e.i-iv), and a distinct ladder pattern on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.e) were observed in 0.8 and 1.0 M, while faded color changes were observed for all dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.e.i-iv) and a weak ladder pattern on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.e) were observed in 0.4 and 0.6 M. Analysing these observations, 0.8 M concentration of betaine was found to be suitable for this PSR amplification. Betaine plays a crucial role as a zwitterionic molecule in balancing reactions to intensify species specificity (Foo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). An inadequate betaine concentration may lead to false positives, as it is unable to inhibit secondary structures, while an unrestricted betaine concentration hinders the successful amplification process (Foo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another study on microbial DNA identification (Milton et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) also showed that 0.8 M betaine effectively minimizes nonspecific amplification without affecting reaction kinetics. A previous study also reported lower concentrations of 0.5 M for identifying chevon (Shree et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause temperature is a crucial factor in PSR amplification, a temperature range of 60\u0026ndash;70\u0026deg;C was analyzed for optimization. In this assessment, no color change was observed for any of the dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A. f. i-iv), and no ladder pattern on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B. f.) were observed at 60\u0026deg;C. But at 61\u0026ndash;63℃, gradual fading of the color change (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A. f. i-iv), and a weaker ladder pattern on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B. f.), were also observed. At a temperature in the 64\u0026ndash;70℃ range, a gradual increase in the intensity of the color change for all dyes was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.f.i-iv) and visible ladder patterns on agarose gel electrophoresis were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.f). Therefore, 65\u0026deg;C was determined as the optimal temperature for the PSR assay.\u003c/p\u003e \u003cp\u003eThe PSR assay was also analyzed over a wide time range of 54\u0026ndash;70 min. No amplification was found in 54 and 56 minutes for both dye-based (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.g.i-iv) and agarose electrophoresis methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.g), but faded results were observed after 58 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.g.i-iv and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.g). The stronger intensity of the color shift of the dyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A.g.i-iv), and visible ladder patterns on gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B.g) was consistent between 60 and 70 min. Based on these observations, 60 min was selected as the optimal reaction time for the PSR assay.\u003c/p\u003e \u003cp\u003eThus, for this PSR assay protocol study, a temperature of 65\u0026deg;C in association with an incubation time of 60 min was selected as a standardized combination to obtain successful amplification without any hindrance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Specificity check\u003c/h2\u003e \u003cp\u003eThe novel species-specific 12S rRNA primers for both PCR and PSR assays for ducks demonstrated accurate specificity with positive results in confined lanes of duck-specific samples. In the PSR assay, various dyes were applied for naked-eye detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A.i-iv), and agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B.i) showed that reactions consisting of DNA samples of chicken, quail, turkey, cattle, buffalo, sheep, goat, pig, and negative control were not amplified. The repeatability of the developed PSR assay was established by repeating the test three times. These observations correspond to the PCR assay results performed with species-specific 12S rRNA primers for ducks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B.ii) and ensured high specificity for duck DNA detection. The high specificity and repeatability of the PSR assay have been reported for various target species, such as pork (Danawadkar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), chevon (Shree et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), buffalo (Jawla and Chatli, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and cattle (Jawla et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The use of an easier primer design method and the utilization of only a pair of primers at low concentrations enable higher specificity in PSR (Liu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) by reducing nonspecific amplification through accurate hybridization with the target DNA compared to the LAMP assay (Li and Fan, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Sensitivity check\u003c/h2\u003e \u003cp\u003eTo validate the limit of detection (LOD), the sensitivity of the PSR assay was analyzed by naked-eye visualization through dye-based detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A.i-iv), and agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B.i). In this naked-eye visualization analysis, a gradual decrease in the dye efficiency occurred with tenfold serial dilution of DNA concentrations (from 30 ng) for ten times, with non-traceability of the PSR end-point product below 3 femtograms (fg) concentration. With this LOD, the PSR assay showed a much higher sensitivity of up to 3 fg. Thus, the PSR assay efficiency to detect the DNA was three times that of tenfold further serial dilution in comparison to the PCR assay efficiency, which showed a sensitivity of only 3 picogram (pg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B.ii). The repeatability of the developed PSR assay was established by repeating the test three times. The PSR assay, with a LOD of 3 fg, outperforms the sensitivity of various reported nucleic acid-based amplification methods in terms of the efficient detection of duck meat. While in comparison to the PSR assay, the multiplex PCR assay has an LOD of 0.01 ng (Yang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), LAMP has LODs of 10 pg (Cho et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), real-time RPA has shown an LOD of 10 ng DNA, and the RPA lateral flow strip assay has an LOD of 1 pg (Zhou et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Authentication/Validation of PSR assay\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Heat-treated samples at different temperatures and times\u003c/h2\u003e \u003cp\u003eThe developed PSR assay was employed to detect duck content in heat-treated or boiled duck meat products by extracting DNA from duck meat, as described in section 2.9.1. A total of 30 samples at different time and temperature variations were used to validate the efficacy of the PSR assay over a wide range using naked-eye detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A.i-iv), and agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.B). All 30 samples showed successful detection of duck DNA over this range, with no amplification observed in the negative control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe application of heat while preparing processed meat may enhance the disruption of the cell membrane and aid in DNA extraction and subsequent amplification efficacy (Arun et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), making DNA detection-based methods more reliable for identifying adulteration in processed food. In the current study, successful amplification of heat-treated samples at temperatures of up to 121℃ for 60 min was observed. This indicates that the PSR assay is efficacious even after high processing temperatures, making it a suitable method for identifying duck content in heat-treated or processed commercial meat products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Processed, cooked, and frozen samples\u003c/h2\u003e \u003cp\u003eThe PSR assay was also used to detect duck content in processed, cooked, and frozen duck meat products. DNA was extracted from duck meat, as described in Section 2.9.2. The sample size was kept at 20 for different variations of cooking or freezing procedures, varying with time and temperature combinations. To validate the efficiency of the PSR assay over a wide range, we used dye-based visual detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.A.i-iv), and agarose gel electrophoresis methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.B) were performed. All 20 samples showed successful detection of duck DNA over this range, with no amplification observed in the negative control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCR detection can be hindered by the addition of spices, salt, high-heat processing, and cooking oil, which may lead to potential inaccuracies (Yang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, when the PSR assay was tested on such samples, it was found to be reliable and sensitive for detecting duck meat, making it more suitable for on-site detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 Different combinations of binary meat admixture\u003c/h2\u003e \u003cp\u003eDuck meat was mixed with sheep meat in a series of combinations to prepare binary meat admixture as described in section 2.9.3, to validate the highly sensitive and species-specific detection nature of the PSR assay. For this, standardized naked-eye detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.A.i-iv), and agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.B) were applied to confirm the suitability of the PSR assay with a minimum LOD of 0.01%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn previous studies, multiplex PCR reported a 1% LOD (Hou et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), fluorescence-based RPA at a 1% LOD (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and PCR\u0026thinsp;+\u0026thinsp;FITC/biotin-labeled- primers with a lateral flow strip (LFS) at a 0.05% LOD (Qin et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) identified the duck content in the designed experimental adulteration ratio. However, this PSR assay study has yielded positive testing results, even with a 0.01% LOD, which surpasses all other methods in detecting adulteration to this extent.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eFor the first time, a 12S rRNA gene PSR-based assay was designed for the rapid, accurate, and sensitive detection of duck meat in adulterated meat samples of other species where duck tissue has been added as an adulterant. The developed PSR assay demonstrated the capability of detecting as low as 3 fg/\u0026micro;L of duck genomic DNA within 60 min at 65\u0026deg;C. The PSR assay exhibited high specificity, with no cross-reactivity with the eight other meat species tested. The assay can identify duck meat adulteration at levels as low as 0.01% in sheep meat mixtures. The PSR assay successfully detected duck DNA in 30 combinations of heat-treated samples (up to a maximum of 121\u0026deg;C for 60 min) and 20 processed or frozen samples, demonstrating its robustness across various sample types. Naked-eye detection using different dyes, such as HNB, Acridine orange, SYBR Gold, and SYBR Green I, enables direct visualisation of the results. Overall, the most precise result was obtained by SYBR Green I. However, for HNB dye detection, UV light is not required, making it more suitable for point-of-care testing. The PSR method surpasses PCR and other techniques in terms of sensitivity, rapidity, and simplicity for on-site detection. The developed PSR assay exhibits high sensitivity, specificity, and suitability for point-of-care testing, making it potentially valuable for regulatory agencies and food producers to rapidly screen for meat adulteration when duck meat is used as an adulterant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they do not have any competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.C.: Writing \u0026ndash; original draft, writing \u0026ndash; review and editing, investigation and formal analysis, visualization and validation. S.S.: Writing \u0026ndash; review and editing, conceptualization, methodology, visualization and validation, project administration, supervision, and funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to SERB (ANRF sanction order No. SUR/2022/002154) for funding the research on the use of PSR-based isothermal amplification for detecting meat adulteration.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper. Any other data, if required will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArun \u0026Ouml;\u0026Ouml;, Muratoğlu K, Eker FY (2014) The effect of heat processing and pH on PCR detection of genetically modified (GM) soy in meat products. 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Food Chem Mol Sci 6:100162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fochms.2023.100162\u003c/span\u003e\u003cspan address=\"10.1016/j.fochms.2023.100162\" 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":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adulteration, Duck meat, on-site detection, PSR assay, rapid detection, 12S rRNA gene","lastPublishedDoi":"10.21203/rs.3.rs-8627709/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8627709/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMeat adulteration is a significant global concern for the food industry, compromising food safety by substituting higher-value meat with lower-cost alternatives. Duck meat is frequently used to adulterate other, more expensive commercial meats due to its similar texture and appearance. This study aimed to develop a polymerase spiral reaction (PSR)-based assay for the rapid detection of duck meat in meat samples of other species adulterated with duck tissue. The 12S rRNA duck gene was targeted for PSR-based amplification. The optimized assay was completed in 60 min when the temperature was maintained at 65\u0026deg;C, resulting in the successful detection of Duck DNA with a sensitivity of up to 3 fg/\u0026micro;L of genomic DNA. The study showed no cross-reactivity with eight other meat species and detected up to 0.01% duck adulteration in sheep meat admixtures. The PSR assay was able to precisely determine the duck content in 30 heat-treated and 20 frozen/processed samples, thus demonstrating the robustness of the developed assay. This PSR-based detection method using 12S rRNA can provide a rapid, sensitive, and cost-effective approach for the on-site detection of duck meat adulteration.\u003c/p\u003e","manuscriptTitle":"Enhancing Food Authenticity: A Dye-Based Polymerase Spiral Reaction for Rapid Detection of Duck Tissue as Adulterants in Meat Samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 10:07:42","doi":"10.21203/rs.3.rs-8627709/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-08T09:51:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T08:28:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290084542899834134949472580976308837481","date":"2026-01-30T08:29:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-29T09:11:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T07:33:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T07:33:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Analytical Methods","date":"2026-01-17T18:01:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"793bf863-3859-49ca-a64c-5148c2c1a671","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T09:24:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 10:07:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8627709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8627709","identity":"rs-8627709","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}