{"paper_id":"01ccd9b9-632d-4e8d-9a5d-58d380182cae","body_text":"A highly sensitive multiplex lateral flow immunoassay for simultaneous detection of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7 | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A highly sensitive multiplex lateral flow immunoassay for simultaneous detection of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7 Dianbo Zhao, Jialei Liu, Juan Du, Kai Liu, Yanhong Bai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2073305/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 In this study, a sensitive, fast and reliable multiplex lateral flow immunoassay based on multiple PCR and gold nanoparticles (AuNPs) was developed. Genomic DNA of Listeria monocytogenes , Salmonella Typhimurium and Escherichia coli O157:H7 was extracted by a simple boiling method. Three pairs of primers were designed and labeled according to specific gene fragment of the three strains for multiple PCR. The PCR products were then conjugated with AuNPs and detected by multiplex lateral flow strip, on which the test lines loaded with anti-biotin antibody, anti-FITC antibody and anti-digoxin antibody corresponding to the labels of primers, respectively. Results showed the limit of detection of L. monocytogenes , S. Typhimurium and E. coli O157:H7 in pure culture were 1.0×10 1 CFU mL − 1 , 1.0×10 2 CFU mL − 1 and 1.6×10 2 CFU mL − 1 , respectively, without culture enrichment. In addition, the lateral flow immunoassay showed good specificity, no reaction to each other or no cross-reactivity with other tested foodborne bacteria were observed. The detection took less than 4 h including PCR amplification, AuNPs conjugation and strip detection. Furthermore, the developed method was applied for the detection of food samples (chicken breast), which was verified by plate count method. The recoveries ranged from 92.7–112.1%, with the coefficient of variation less than 8.73%, revealing the feasible and reliable application of this method in practical sample. Therefore, the developed multiplex lateral flow strip is sensitive, accurate and visualized, which is applicable to simultaneous detection of the three foodborne pathogenic bacteria in food sample. multiplex lateral flow immunoassay multiplex PCR gold nanoparticles foodborne pathogenic bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Foodborne illnesses are usually contagious or toxic and are caused by contaminated food or water. In 2015, WHO estimated that unsafe food cause 600 million foodborne illnesses and 420,000 deaths every year around the world. Foodborne pathogens are the leading cause of foodborne disease outbreaks, which can cause severe diarrhoea or debilitating infections including meningitis. Salmonella , Enterohaemorrhagic Escherichia coli and Listeria are among the most common foodborne pathogens that affect millions of people annually, which are mainly associated with raw meat, eggs, raw milk and poultry products (Wang, 2011 ). Salmonella Typhimurium ( S. Typhimurium) is a mainly species of genus Salmonella , it could cause symptoms like abdominal pain and diarrhoea, fever, nausea and vomiting (Moir, 2010 ; Nagarajan, 2009; Nyachuba, 2010 ; Foley, 2011 ). Escherichia coli O157:H7 ( E. coli O157:H7) is a commonly serotype of Enterohaemorrhagic E. coli , which produce type I and/or type II shiga toxin and cause serious foodborne illnesses (Yang, 2020). Listeria monocytogenes ( L. monocytogenes ) could cause listeriosis, which leads to miscarriage in pregnant women or death of newborn babies. The mortality rate of listeriosis can be as high as 30% (Choi, 2018). Therefore, a highly sensitive and accurate detection method in food is crucial for preventing foodborne illnesses caused by these pathogenic bacteria. The current gold standard for the detection of these pathogens is conventional culture-based methods that is time consuming and labor intensive (Yang, 2013). Polymerase chain reaction (PCR)-based methods have been widely used for the rapid, sensitive and specific detection of pathogenic bacteria in foods (Bhaduri, 2001 ; Bonetta, 2011; Cadirci, 2010; Wang, 2007). Furthermore, multiplex PCR (Gilbert, 2003; Morin, 2004; Park, 2006 ) allows simultaneous detection and amplification of more than one gene in one reaction (Lee, 2014). It provides a simple and sensitive tool for the simultaneous detection of multiple pathogenic bacteria. However, the PCR-based assays require complicated gel electrophoresis and gel imaging systems to obtain the detection results (Yang, 2017), which limits its application in easy-to-use and rapid detection. In recent years, lateral flow immunoassay (LFIA) is a promising detection method and has been widely used in various fields such as food safety detection, clinical diagnosis, environmental analysis, drug residue analysis owing to its advantages of user-friendly operation, rapid, cost-effective visualized and does not rely on professional techniques or instruments (Tian, 2021). Gold nanoparticles (AuNPs) is widely used as labeling material in LFIA due to its excellent optical property, good biocompatibility and easy synthesis (Hou, 2020). The routine single target test of a single sample does not completely prevent the food safety problems caused by pathogenic bacteria. It is necessary to develop a rapid and sensitive method for simultaneously detecting of various pathogenic bacteria. Meanwhile, the multiplex detection of the pathogenic bacteria are time and cost saving, and the requirement of sample and labor could be reduced. Herein, a novel LFIA based on multiplex PCR for simultaneous detection of S. Typhimurium, E. coli O157:H7 and L. monocytogenes was developed. The sensitivity of PCR system was improved by screening the concentration of primers and annealing temperature. AuNPs was prepared and conjugated with PCR products under acid environment to trace and amplify the signal of PCR product. The AuNP-PCR product was visualized detected by antibodies on LFIA strip and shows red color, and no color will be appeared on the strip if thiere is no target bacterium in sample, which is more convenient and accurate than the indirect competitive method. To the best of our knowledge, it is the first report of multiplex PCR based LFIA for simultaneous detection of S. Typhimurium, E. coli O157:H7 and L. monocytogenes . This study offers a promising approach for rapid, sensitive and accurate detection of foodborne pathogens. 2. Materials And Methods 2.1 Materials L. monocytogenes (ATCC 15313), E. coli O157:H7 (ATCC 43895), P. aeruginosa (ATCC 10145), V. parahaemolyticus (ATCC 17802) and S. aureus (ATCC 6538) were employed from American Type Culture Collection (ATCC), S. Typhimurium (CICC 21484) was employed from China Center of Industrial Culture Collection (CICC). Thiol (SH) labeled primer LM- hly F, biotin labeled primer LM- hly R specifically to L. monocytogenes ; SH labeled primer ST- hut F, FITC labeled primer ST- hut R specifically to S. Typhimurium; and SH labeled primer EC- rfbE F, digoxin labeled primer EC- rfbE R specifically to E. coli O157:H7, were procured from BGI Technology Co., Ltd (Shenzhen, China). Sodium citrate, Taq PCR Master Mix, Ezup column bacteria genomic DNA isolation kit, DNA marker, and 4S Green Plus nucleic acid stain were procured from Sangon Biotechnology Co., Ltd (Shanghai, China). Agarose was procured from Solarbio Life Sciences (Beijing, China). Anti-biotin antibody was procured from Abcam (Shanghai, China); Anti-FITC antibody was procured from BioMag beads (Wuxi, China); Anti-digoxin was procured from Jackson (USA). Backing card, sample pad, NC membrane and absorbent pad were procured from Shanghai JieYi Biotechnology Co., Ltd (Shanghai, China). Chloroauric acid was procured from Macklin (Shanghai, China). Trypticase soy broth (TSB) was bought from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, China). Commercial test kit was bought from Meizheng Bio (Beijing, China). 2.2 Bacterial culture S. Typhimurium, E. coli O157: H7, L. monocytogenes , S. aureus , V. parahaemolyticus and P. aeruginosa were cultured in TSB and incubated at 37℃ with shaking (150 rpm). The logarithmic phase cultures were collected through centrifugation at 7,512 Xg for 2 min, followed by washing and resuspending in sterile pH 7.4 phosphate buffer (PBS, 0.01 M). Then, the bacteria cultures were diluted with sterile PBS to make different concentrations. The number of bacteria was determined by plate count method. 2.3 Genomic DNA isolation and multiplex PCR Genomic DNA of the bacteria was extracted by simple boiling for 10 minutes, after cooling down to room temperature and centrifuged at 7,512 Xg for 2 min, the supernatant which contained DNA was used as template for multiplex PCR. Meanwhile, DNA was extracted and purified according to the manufacturer's protocol of Ezup column bacteria genomic DNA isolation kit to determine the limit of detection of pure DNA. The quantity and quality of the obtained DNA were evaluated according to A260 and A260/A280, respectively. Three pairs of primers specific to the corresponding bacteria were used in the multiplex PCR assay (Table S1). The multiplex PCR was performed with reaction solution contained 2 µL of each genomic DNA, 2 µL of each primer (at a final concentration of 0.8 µM), 25 µL 2×Taq PCR Master Mix buffer and sterilized ddH 2 O to make final volume of 50 µL. PCR reactions were carried out using a S1000TM thermal cycler (BioRad Laboratories, USA), the PCR condition included initial denaturation for 5 min at 95°C, followed by 32 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 30 s, and then a final extension at 72°C for 10 min. PCR products were stored at -20°C for further detection. 2.4 Conjugation of AuNPs with multiplex PCR products The AuNPs was prepared by the Turkevich method (Kimling, 206) using sodium citrate as reducing agent with slightly modifications. Briefly, 100 µL of 0.1 mM HAuCl 4 was added to 99.9 mL Milli Q water and stirred vigorously. After boiling, 1 mL of 1% sodium citrate was added quickly to the HAuCl 4 solution under stirring and boiling for another 10 min. The obtained red color solution will then cool to room temperature and stored at 4°C in dark. The synthesized AuNPs were characterized by UV-vis spectrum, TEM and FT-IR analysis. PCR products were conjugated with AuNPs in a acid environment, specifically, 5 µL of 1 M citric acid buffer (PH 3) was added to 50 µL PCR products, then 50 µL AuNPs was transferred to the above solution and incubated for 3 min to make AuNP-PCR product conjugation. Afterward, the mixture was centrifuged at 4694 Xg for 5 min, the pellet was resuspended in 100 µL of 10% BSA for blocking, then the AuNP-PCR product-BSA composite was obtained. 2.5 Fabrication of LFIA The LFIA was prepared according to previous study (Wu, 2021), it consisted of four parts: sample pad (1.5 cm × 3 mm), NC membrane (2.5 cm × 3 mm), absorbent pad (3 cm × 3 mm) and backing pad (7 cm × 3 mm). Totally three test lines on the NC membrane was designed for the detection of the corresponding three strains. First test line (T1) was spotted with anti-biotin antibody, second test line (T2) was spotted with anti-FITC antibody and the third test line (T3) was spotted with anti-digoxin antibody at a rate of 1 µL cm − 1 , respectively. Finally, the LFIA was dried at 37°C for 4 h and then cut into 3 mm width, and further stored in a desiccator kept in dark. 2.6 Multiplex LFIA procedure for the detection of S. Typhimurium, E. coli O157: H7 and L. monocytogenes A series of concentrations of S. Typhimurium, E. coli O157: H7 and L. monocytogenes ranged from 10 9 CFU mL − 1 to 10 1 CFU mL − 1 were prepared to determine the limit of detection of the assay. After DNA extraction and PCR amplification, 20 µL of the AuNP-PCR product-BSA suspension were detected by LFIA and results were observed in 10 minutes. As control, PCR products were analyzed using 2% agarose gel electrophoresis and observed under Gel Imager System (geldoc EZ, USA). In order to confirm the specificity of this assay, three common foodborne pathogens at a high concentration (10 7 CFU mL − 1 ) were detected by the LFIA, including S. aureus , V. parahaemolyticus and P. aeruginosa . All experiments were repeated three times to prove credibility. 2.7 Detection of artificially contaminated food samples Chicken breast were obtained from a local supermarket and analyzed within 1 h after purchase. Samples were pretreatment according to the culture-based method. Briefly, chicken breast was divided into 3 portions of 25g and washed twice with deionized water, soaked in 75% ethanol for 10 min, and then sterilized with UV light for 30 min. Bacteria in varying concentrations were spread evenly each 25 g chicken breast and incubated overnight respectively. Then, the chicken breast was placed in 225 mL PBS, and treated with flapped and concussed to obtain the bacterial suspension and detected by the proposed method. At the same time, the plate count method was performed to verify the detection. 3. Results And Discussion 3.1 The detection principle of multiplex LFIA The principle of the developed detection method of bacteria was demonstrated in Fig. 1 . AuNPs in red wine color was prepared by using sodium citrate as reducing and stabilizing agent. Bacterial DNA was extracted by boiling for 10 minutes, which is simple, rapid, economic and environmental friendly as compare with traditional solvent extraction and commercial kits. For multiplex PCR, three pairs of primers specific for hut gene of S. Typhimurium, rfbE gene of E. coli O157: H7 and hly gene of L. monocytogenes were prepared. The forward primers for hut , rfbE and hly gene were labeled with SH group, and the reverse primers for hut , rfbE and hly gene were labeled with FITC, digoxin and biotin, respectively. As a result, multiplex PCR products contained both SH group and FITC amplified from S. Typhimurium DNA, contained both SH group and digoxin amplified from E. coli O157: H7 DNA, and contained both SH group and biotin and amplified from L. monocytogenes DNA. The PCR products were then conjugated with AuNPs in citric acid buffer (pH 3), and the remaining sites on AuNPs were blocked by BSA. The AuNP-PCR product-BSA was detected by LFIA strip by adding it to sample pad and migrated along the LFIA toward absorbent pad. When it reached NC membrane, biotin would be captured by anti-biotin antibody on T1, FITC would be captured by anti-FITC antibody on T2, and digoxin would be captured by anti-digoxin antibody on T3. If samples contained target bacteria, the corresponding T lines would appear red color, which was attributed to the AuNPs. With the accumulation of PCR products, the red bands on the T line would be gradually deepened. On the contrary, no red band would appear on the T line if target bacteria were absent in the sample. 3.2 Optimization of multiplex PCR assay and antibody concentration on test line of LFIA In order to ensure the specific of the multiplex PCR and obtain each specific PCR product, annealing temperature (48.8℃, 50℃, 53℃, 55.5℃, 56.2℃, 58.8℃, 60℃, 62.2℃) and concentration of primer pairs (Table S2) were optimized in this study. The multiplex PCR products were 360 bp, 495 bp and 678 bp, corresponding to specific genes of L. monocytogenes , S. Typhimurium and E. coli O157:H7, respectively (Fig. 2 A and 2 B). The gray value of the band brightness was measured and analyzed by image J software. It was found that the band brightness reached the maximum when the annealing temperature was 53℃ (Fig. 2 C) and the concentrations of the primer pairs was 0.8 µM respectively (Fig. 2 D). The antibody concentration on T line is critical for the detection and the cost of LFIA. The anti-biotin antibody, anti-FITC antibody and anti-digoxin antibody at the concentration of 0.4M, 0.6M, 0.8M and 1M were optimized respectively. It was found that red bands reached the maximum when the concentrations of all three antibodies were 1M (Fig. 2 E). The intensity of red bands analyzed by image J software were further confirmed the optimal concentration of all three antibodies is 1M (Fig. 2 E insert image). Therefore, the annealing temperature at 53℃ and the concentrations of all three pairs of primers at 0.8 µM, and all three antibodies at concentration of 1M were used as optimal conditions for the detection. 3.3 Characterization of AuNPs and AuNP@PCR products The AuNPs were synthesized by HAuCl 4 and sodium citrate through heating stirring process. As demonstrated in Fig. 3 , the AuNPs were uniformly elliptical spherical and the size were approximately 50–75 nm, with maximum absorption peak at 525 nm.. The size of AuNP@PCR products were increased to approximately 100–150 nm,. Furthermore, the zeta potential of AuNPs and AuNP@PCR products were − 26.4 ± 1.2 mV and − 37.75 ± 1.5 mV, respectively (Fig. 3 F), indicating the AuNPs was successfully conjugated with PCR products. According to the FTIR spectra of AuNPs, peaks at 1249, 1452 and 3240 were due to C-CH3 tensile vibration and the bending vibration of methylene C-H, O-H stretching vibration of hydroxyl functional groups, respectively (Sivakumar, 2009; Sharma, 2012; Ganapuram, 2015; Tanzil, 2016; Babu. 2011; Babu, 2012), the FTIR spectra of AuNP@PCR products showed more peaks than AuNPs, due to the existent of DNA, biotin, FITC digoxin, etc, its functional groups are more complex. 3.4 Sensitivity of the assay The limit of detection of genomic DNA by the LFIA strip was firstly studied. Serial dilutions (70, 35, 17.5, 8.8, 4.4, 2.2, 1.1, 0.6, 0.3, 0.15 and 0.075 ng/µL) of each bacteria's DNA was prepared. The DNA were was subjected to multiplex PCR assay, and sterilized ddH 2 O was performed as control in the same manner. After multiplex PCR amplification, PCR products were conjugated with AuNPs and then detected by LFIA. The PCR products were analyzed using 2% agarose gel electrophoresis and observed under Gel Imager System (geldoc EZ, USA) as control. The limit of detection of genomic DNA were found to be 150 pg/µL for L. monocytogenes ,150 pg/µL for S. Typhimurium, and 600 pg/µL for E. coli O157:H7 (Fig. 4 A and 4 B). The limit of detection of DNA examined by traditional agarose gel method were 1.1 ng/µL for L. monocytogenes , S. Typhimurium and E. coli O157:H7 (Fig.S1A), which is less sensitive than the LFIA proposed by this study. The detection sensitive for the three bacteria of the developed method was studied under the optimized conditions. Different concentrations of L. monocytogenes , S. Typhimurium and E. coli O157:H7 (10 1 -10 9 CFU mL − 1 ) were prepared by diluting the logarithmic phase bacteria with sterile PBS, and quantified by plate count method. Results showed that the limit of detection of multiplex PCR products for L. monocytogenes , S. Typhimurium and E. coli O157:H7 by traditional agarose gel were 0.9×10 7 , 1.2×10 7 , 1.5×10 7 CFU mL − 1 , respectively (Fig.S1B). As demonstrated in Fig. 4 C, the detection limit of E. coli O157:H7, S. Typhimurium and L. monocytogenes were 1.6×10 2 CFU mL − 1 , 1.0×10 2 CFU mL − 1 , 1.0×10 1 CFU mL − 1 by the LFIA with naked eye. The detection sensitivity of the LFIA proposed in this study was improved 10 6 CFU mL − 1 for L. monocytogenes and 10 5 CFU mL − 1 for S. Typhimurium and E. coli O157:H7, as compared with traditional multiplex PCR assay. This improvement is due to AuNPs signal amplification and LFIA convenient and fast detection, whereas agarose gel is time-consuming, toxic, and requires gel imager equipment. Moreover, according to the analysis by Image J, intensity of T lines has a good linear relationship with the concentration of corresponding bacteria. The linear equation of L. monocytogenes concentration in the range of 1.0×10 1 to 0.8×10 9 CFU mL − 1 was Y = 33.29 + 32.50 X with correlation coefficient (R 2 ) of 0.941 (Fig. 4 D), S. Typhimurium concentration in the range of 1.0×10 2 to 1.1×10 9 CFU mL − 1 was Y= -74.08 + 57.02 X with correlation coefficient (R 2 ) of 0.996 (Fig. 4 E), E. coli O157:H7 concentration in the range of 1.6×10 2 to 1.5×10 9 CFU mL − 1 was Y= -64.36 + 43.91X with correlation coefficient (R 2 ) of 0.962 (Fig. 4 F). The multiplex LFIA employed PCR amplification and AuNPs labeling signal to improve the sensitivity of the detection of foodborne pathogenic bacteria. By comparing with some recently reported PCR-based methods and AuNP-based methods for detection of foodborne pathogenic bacteria, the proposed method showed better performance in terms of sensitivity and rapid (Table 1 ). The developed multiplex strips are faster, more sensitive and less time consuming than single T line strip when detecting of three species of bacteria. Table 1 Comparison of reported multiplex detection methods for target bacteria Detection method Target bacteria Detection limit Total time Reference Antibody-AuNP-MNPs SA SE, EC EC, YE, ST, LM LM, ST, EC LM, ST, EC LM, ST, EC 1.5×10 3 CFU/mL > 24 h (Sung,2013) 7-plex PCR 3 CFU/10g > 16 h (Wang, 2011 ) Chemiluminescence-IMS real-time PCR-SEL 10 4 CFU/mL 10 CFU > 18 h > 21 h (Magliulo, 2007) (Wang, 2013 ) IMS-PMA-mPCR 10 2 CFU/mL 4.5 h (Yang, 2013) mPCR 10 2 CFU/mL 4 h This work 3.5 Specificity of the assay The multiplex LFIA was tested for the specificity against three other foodborne pathogen bacteria including S. aureus , V. parahaemolyticus and P. aeruginosa . The traditional agarose gel verified the accurate and specific of muplex PCR (Fig.S1C). As demonstrated in Fig. 5 A and B, the appearance of red band on the separate T line was only observed when corresponding target bacteria is present. No significant changes were observed in the presence of other disturbing foodborne pathogen bacteria. Therefore, the multiplex LFIA is highly specific and ensures accurate detection of target bacteria. 3.6 Detection of bacteria in food samples The prepared chicken breast samples spiked with E. coli O157:H7 (10 1 -10 8 CFU mL − 1 ), S. Typhimurium (10 1 -10 8 CFU mL − 1 ) and L. monocytogenes (10 1 -10 8 CFU mL − 1 ) were tested by the multiplex LFIA strip, the recovery rates and their coefficients of variation were calculated, and the accuracy of the proposed method was evaluated by plate count method. Traditional agarose gel method was also used to detect the multiplex PCR products as control, and the limit of detection turned out to be 0.5×10 7 , 1.9×10 7 , 3.7×10 7 CFU mL − 1 of L. monocytogenes , S. Typhimurium and E. coli O157:H7, respectively (Fig.S1D). For comparison, a commercial L. monocytogenes LFIA strip, S. Typhimurium LFIA strip and E. coli O157:H7 LFIA (Meizheng Bio, China) were employed to detect L. monocytogenes , S. Typhimurium and E. coli O157:H7, respectively. The limit of detection were 9.2×10 7 CFU mL − 1 , 1.2 ×10 6 CFU mL − 1 , 1.6 ×10 6 CFU mL − 1 of L. monocytogenes , S. Typhimurium and E. coli O157:H7, respectively (Fig.S2). Therefore, the developed multiplex LFIA assay in this study is more sensitive and more efficient as compare with commercial kit. As demonstrated in Fig. 5 C, the limit of detection of the developed multiplex LFIA strip of L. monocytogenes , S. Typhimurium and E. coli O157:H7 was 0.3×10 1 CFU mL − 1 , 2.0×10 1 CFU mL − 1 and 3.5×10 2 CFU mL − 1 , respectively. As compare with commercial LFIA strips, the limit of detection of developed multiplex LFIA strip was improved 10 6 CFU mL − 1 of L. monocytogenes , 10 5 CFU mL − 1 of S. Typhimurium and 10 4 CFU mL − 1 of E. coli O157:H7, and the result can be read in one strip, which is convenient and low cost. The intensity of red band on T lines were gradually increased with the increasing of bacteria concentration (Fig. 5 D-F). The recoveries of this study were ranging from 92.7–112.1%, with the coefficient of variation less than 8.73%, and the recoveries of plate count were in the range of 96.6–102.9% with the coefficient of variation less than 7.28% (Table 2 ). Therefore, the developed multiplex LFIA is sensitive and accurate, and shows a promising application in simultaneous detection of foodborne pathogen bacteria in food samples. Table 2 Analytical results of bacteria spiked in chicken breast by this detection system and using plate counting (n = 3). Spiked (lgCFU/mL) Mean found ± SD (lgCFU/mL) Recovery (%) CV (%) This method Plate counting This method Plate counting This method Plate counting LM 4.70 4.70 ± 3.19 4.71 ± 3.07 99.4 102.9 3.11 2.28 5.70 5.70 ± 4.23 5.69 ± 4.14 100.9 98.0 3.36 7.28 6.70 6.73 ± 5.42 6.69 ± 5.15 106.7 97.5 4.92 2.90 ST 5.28 5.30 ± 3.92 5.26 ± 3.70 105.2 96.6 4.17 2.72 6.28 6.29 ± 4.82 6.28 ± 4.66 103.6 99.5 3.38 2.40 7.28 7.33 ± 6.27 7.29 ± 5.55 112.1 102.1 8.73 1.84 EC 5.58 5.55 ± 4.13 5.58 ± 3.97 92.9 99.7 3.81 2.44 6.58 6.55 ± 5.27 6.58 ± 4.26 92.7 100.7 5.23 0.47 7.58 7.60 ± 6.27 7.57 ± 5.35 104.8 97.4 4.69 0.60 4. Conclusion In the present study, a novel multiplex LFIA was developed for simultaneous detection of L. monocytogenes , S. Typhimurium and E. coli O157:H7 by naked eye. The highly sensitive of this method was take advantages of PCR amplification and AuNPs labeling. And benefit by the specific of primer and antibody, this method was highly reliable. The detection limitation of L. monocytogenes , S. Typhimurium, E. coli O157:H7 was 1.0×10 1 CFU mL − 1 , 1.0×10 2 CFU mL − 1 and 1.6×10 2 CFU mL − 1 , respectively. The assay took 4 h including 1.5 h for multiplex PCR and 2 h for preparing AuNP@PCR product@BSA, and finally LFIA strip detection. Thus, the developed method was fast, sensitive, specific and reliable for the detection of the three target bacteria in food sample, and could be applied for simultaneous detection of L. monocytogenes , S. Typhimurium, E. coli O157:H7. Declarations Acknowledgments: This work was funded by the major special project on public welfare of Henan Province [grant number 201300110100]. Compliance with Ethical: This manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. Funding: This work was funded by the major special project on public welfare of Henan Province [grant number 201300110100]. Conflict of Interest : The authors declared that they have no conflicts of interest. Author contributions : Dianbo Zhao: Conception, Methodology, Writing-review. Jialei Liu: Material preparation, Data collection, Writing-original draft. Juan Du: Analysis and Writing-editing. Kai Liu: Material preparation. Yanhong Bai: Funding acquisition, Resources, Supervision. All authors read and approved the final manuscript. Ethical approval : This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent: Informed consent was obtained from all individual participants included in the study. D ata availability statemen t The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Babu PJ, Sharma P, Kalita MC. (2011). Green synthesis of biocompatible gold nanoparticles using Fagopyrum esculentum leaf extract. 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Journal fur Verbraucherschutz und Lebensmittelsicherheit-Journal of Consumer Protection and Food Safety, 6(4), 441–447. https://doi:10.1007/s00003-011-0696-1. Wang L, Li Y, Mustaphai A. (2007). Rapid and simultaneous quantitation of Escherichia coli O157:H7, Salmonella , and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. Journal of Food Protection, 70(6), 1366-1372. https://doi:10.4315/0362-028X-70.6.1366. Wang YX, Suo B. (2013). Evaluation of a multiplex selective enrichment broth SEL for simultaneous detection of injured Salmonella , Escherichia coli O157:H7 and Listeria monocytogenes . Brazilian Journal of Microbiology, 44(3), 737-742. https://doi:10.1590/S1517-83822013000300011. Wu SJ, Du J, Bai YH. (2021). Solvothermal synthesis of α-Fe 2 O 3 polyhedrons and its application in an immunochromatographic strip test for the detection of foodborne pathogen Listeria monocytogenes. 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Supplementary Files FigS1Auncroppedgelsimages.tif FigS1Buncroppedgelsimages.jpg FigS1Cuncroppedgelsimages.jpg FigS1Duncroppedgelsimages.tif Fig2Auncroppedgelsimages.jpg Fig2Buncroppedgelsimages.jpg SupportingInformation.docx 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 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-2073305\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":138671677,\"identity\":\"c01f3d06-d632-4819-8d73-9492634108fb\",\"order_by\":0,\"name\":\"Dianbo Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhengzhou University of Light Industry\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Dianbo\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"},{\"id\":138671678,\"identity\":\"7f84d7c6-6580-4bbe-951f-7db24aa5f8c0\",\"order_by\":1,\"name\":\"Jialei Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhengzhou University of Light Industry\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jialei\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":138671679,\"identity\":\"59885aa6-9c97-4dd3-91a2-d7eec8403cf8\",\"order_by\":2,\"name\":\"Juan Du\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhengzhou University of Light Industry\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Juan\",\"middleName\":\"\",\"lastName\":\"Du\",\"suffix\":\"\"},{\"id\":138671680,\"identity\":\"7637bfdd-515f-430e-b705-b570118d7775\",\"order_by\":3,\"name\":\"Kai Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhengzhou University of Light 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detection\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/cbc46a046dadc474ac2d8ca4.png\"},{\"id\":27025806,\"identity\":\"6d6b4f74-9d46-4944-afe4-353270ce7c0d\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 13:57:58\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":982709,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eOptimization of multiplex PCR annealing temperature (A and C), concentrations of the primer pairs (B and D), and optimization of antibody concentration (E)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/713df6575eb5dd13a228b4a9.png\"},{\"id\":27025804,\"identity\":\"173072ce-06e5-4595-8c3a-12a7e811df4a\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 13:57:58\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":709047,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTEM images of AuNPs (A) and AuNP@PCR products (B), size distribution (C) and UV-visible absorption spectra (D), FT-IR spectrum (E) and zeta potential diagram (F) of AuNPs and AuNP@PCR products.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/81d4957682ab1545e3d036f2.png\"},{\"id\":27025805,\"identity\":\"b3c73fd0-e6c1-4586-882f-188ae60da95e\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 13:57:58\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":859074,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSensitivity of multiplex LFIA for DNA detection (A) and intensity analysis of test lines (B), sensitivity for target bacteria cultures (C), the linear plot between T1 intensity and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e concentration (D), the linear plot between T2 intensity and \\u003cem\\u003eS. \\u003c/em\\u003eTyphimurium (E), and the linear plot between T3 intensity and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7\\u003cem\\u003e \\u003c/em\\u003e(F).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/ebecb51bc5493ad6c112ff9f.png\"},{\"id\":27027034,\"identity\":\"cf2ab49d-96c7-4521-93a2-d789203b8d5e\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 14:02:58\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":780093,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Specificity of multiplex LFIA and (B) intensity analysis of test lines, (C) sensitivity for chicken breast samples contained target bacteria, (D) the linear plot betweenT1 intensity and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e concentration, (E) the linear plot between T2 intensity and \\u003cem\\u003eS. \\u003c/em\\u003eTyphimurium, and (F) the linear plot between T3 intensity and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/bae85cb0df335e875e11d038.png\"},{\"id\":31356768,\"identity\":\"9db0993e-4c35-4d83-bd2b-bd0e4e90437c\",\"added_by\":\"auto\",\"created_at\":\"2023-01-10 11:14:41\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2029524,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/432ec206-dc7c-4d48-a033-cb8e359eba16.pdf\"},{\"id\":27025814,\"identity\":\"860210b9-9024-4057-8388-e72fa7c43def\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 13:57:59\",\"extension\":\"tif\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":29865310,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigS1Auncroppedgelsimages.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/965e7f5e5a45b4b334c904a1.tif\"},{\"id\":27027719,\"identity\":\"3dd3579e-eef9-4e41-ade8-9112bd5678cb\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 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13:57:59\",\"extension\":\"tif\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":29865310,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigS1Duncroppedgelsimages.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/64eddb5602f121736cf8c0a3.tif\"},{\"id\":27025812,\"identity\":\"6bde5134-8ee0-4ff9-a669-71946c1d61e7\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 13:57:58\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":59905,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig2Auncroppedgelsimages.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/25bd4f92154bec9d2c3f30e6.jpg\"},{\"id\":27025810,\"identity\":\"dce9a6b4-2405-4604-8868-75c929343197\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 13:57:58\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":69753,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Fig2Buncroppedgelsimages.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/fdd591eb3c3cedb57728e2e5.jpg\"},{\"id\":27027035,\"identity\":\"03b9d4dd-1a46-43e9-aed1-b8874dd91854\",\"added_by\":\"auto\",\"created_at\":\"2022-09-27 14:02:58\",\"extension\":\"docx\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":660355,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupportingInformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2073305/v1/f1ee9e7546e37e0d7db9070c.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"A highly sensitive multiplex lateral flow immunoassay for simultaneous detection of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eFoodborne illnesses are usually contagious or toxic and are caused by contaminated food or water. In 2015, WHO estimated that unsafe food cause 600\\u0026nbsp;million foodborne illnesses and 420,000 deaths every year around the world. Foodborne pathogens are the leading cause of foodborne disease outbreaks, which can cause severe diarrhoea or debilitating infections including meningitis. \\u003cem\\u003eSalmonella\\u003c/em\\u003e, Enterohaemorrhagic \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e and \\u003cem\\u003eListeria\\u003c/em\\u003e are among the most common foodborne pathogens that affect millions of people annually, which are mainly associated with raw meat, eggs, raw milk and poultry products (Wang, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). \\u003cem\\u003eSalmonella\\u003c/em\\u003e Typhimurium (\\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium) is a mainly species of genus \\u003cem\\u003eSalmonella\\u003c/em\\u003e, it could cause symptoms like abdominal pain and diarrhoea, fever, nausea and vomiting (Moir, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e; Nagarajan, 2009; Nyachuba, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e; Foley, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e O157:H7 (\\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7) is a commonly serotype of Enterohaemorrhagic \\u003cem\\u003eE. coli\\u003c/em\\u003e, which produce type I and/or type II shiga toxin and cause serious foodborne illnesses (Yang, 2020). \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e (\\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e) could cause listeriosis, which leads to miscarriage in pregnant women or death of newborn babies. The mortality rate of listeriosis can be as high as 30% (Choi, 2018). Therefore, a highly sensitive and accurate detection method in food is crucial for preventing foodborne illnesses caused by these pathogenic bacteria.\\u003c/p\\u003e \\u003cp\\u003eThe current gold standard for the detection of these pathogens is conventional culture-based methods that is time consuming and labor intensive (Yang, 2013). Polymerase chain reaction (PCR)-based methods have been widely used for the rapid, sensitive and specific detection of pathogenic bacteria in foods (Bhaduri, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e; Bonetta, 2011; Cadirci, 2010; Wang, 2007). Furthermore, multiplex PCR (Gilbert, 2003; Morin, 2004; Park, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e) allows simultaneous detection and amplification of more than one gene in one reaction (Lee, 2014). It provides a simple and sensitive tool for the simultaneous detection of multiple pathogenic bacteria. However, the PCR-based assays require complicated gel electrophoresis and gel imaging systems to obtain the detection results (Yang, 2017), which limits its application in easy-to-use and rapid detection.\\u003c/p\\u003e \\u003cp\\u003eIn recent years, lateral flow immunoassay (LFIA) is a promising detection method and has been widely used in various fields such as food safety detection, clinical diagnosis, environmental analysis, drug residue analysis owing to its advantages of user-friendly operation, rapid, cost-effective visualized and does not rely on professional techniques or instruments (Tian, 2021). Gold nanoparticles (AuNPs) is widely used as labeling material in LFIA due to its excellent optical property, good biocompatibility and easy synthesis (Hou, 2020). The routine single target test of a single sample does not completely prevent the food safety problems caused by pathogenic bacteria. It is necessary to develop a rapid and sensitive method for simultaneously detecting of various pathogenic bacteria. Meanwhile, the multiplex detection of the pathogenic bacteria are time and cost saving, and the requirement of sample and labor could be reduced.\\u003c/p\\u003e \\u003cp\\u003eHerein, a novel LFIA based on multiplex PCR for simultaneous detection of \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e was developed. The sensitivity of PCR system was improved by screening the concentration of primers and annealing temperature. AuNPs was prepared and conjugated with PCR products under acid environment to trace and amplify the signal of PCR product. The AuNP-PCR product was visualized detected by antibodies on LFIA strip and shows red color, and no color will be appeared on the strip if thiere is no target bacterium in sample, which is more convenient and accurate than the indirect competitive method. To the best of our knowledge, it is the first report of multiplex PCR based LFIA for simultaneous detection of \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e. This study offers a promising approach for rapid, sensitive and accurate detection of foodborne pathogens.\\u003c/p\\u003e\"},{\"header\":\"2. Materials And Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Materials\\u003c/h2\\u003e \\u003cp\\u003e \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e (ATCC 15313), \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 (ATCC 43895), \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e (ATCC 10145), \\u003cem\\u003eV. parahaemolyticus\\u003c/em\\u003e (ATCC 17802) and \\u003cem\\u003eS. aureus\\u003c/em\\u003e (ATCC 6538) were employed from American Type Culture Collection (ATCC), \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium (CICC 21484) was employed from China Center of Industrial Culture Collection (CICC). Thiol (SH) labeled primer LM-\\u003cem\\u003ehly\\u003c/em\\u003eF, biotin labeled primer LM-\\u003cem\\u003ehly\\u003c/em\\u003eR specifically to \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e; SH labeled primer ST-\\u003cem\\u003ehut\\u003c/em\\u003eF, FITC labeled primer ST-\\u003cem\\u003ehut\\u003c/em\\u003eR specifically to \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium; and SH labeled primer EC-\\u003cem\\u003erfbE\\u003c/em\\u003eF, digoxin labeled primer EC-\\u003cem\\u003erfbE\\u003c/em\\u003eR specifically to \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, were procured from BGI Technology Co., Ltd (Shenzhen, China). Sodium citrate, Taq PCR Master Mix, Ezup column bacteria genomic DNA isolation kit, DNA marker, and 4S Green Plus nucleic acid stain were procured from Sangon Biotechnology Co., Ltd (Shanghai, China). Agarose was procured from Solarbio Life Sciences (Beijing, China). Anti-biotin antibody was procured from Abcam (Shanghai, China); Anti-FITC antibody was procured from BioMag beads (Wuxi, China); Anti-digoxin was procured from Jackson (USA). Backing card, sample pad, NC membrane and absorbent pad were procured from Shanghai JieYi Biotechnology Co., Ltd (Shanghai, China). Chloroauric acid was procured from Macklin (Shanghai, China). Trypticase soy broth (TSB) was bought from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, China). Commercial test kit was bought from Meizheng Bio (Beijing, China).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Bacterial culture\\u003c/h2\\u003e \\u003cp\\u003e \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, \\u003cem\\u003eE. coli\\u003c/em\\u003e O157: H7, \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS. aureus\\u003c/em\\u003e, \\u003cem\\u003eV. parahaemolyticus\\u003c/em\\u003e and \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e were cultured in TSB and incubated at 37℃ with shaking (150 rpm). The logarithmic phase cultures were collected through centrifugation at 7,512 Xg for 2 min, followed by washing and resuspending in sterile pH 7.4 phosphate buffer (PBS, 0.01 M). Then, the bacteria cultures were diluted with sterile PBS to make different concentrations. The number of bacteria was determined by plate count method.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Genomic DNA isolation and multiplex PCR\\u003c/h2\\u003e \\u003cp\\u003eGenomic DNA of the bacteria was extracted by simple boiling for 10 minutes, after cooling down to room temperature and centrifuged at 7,512 Xg for 2 min, the supernatant which contained DNA was used as template for multiplex PCR. Meanwhile, DNA was extracted and purified according to the manufacturer's protocol of Ezup column bacteria genomic DNA isolation kit to determine the limit of detection of pure DNA. The quantity and quality of the obtained DNA were evaluated according to A260 and A260/A280, respectively.\\u003c/p\\u003e \\u003cp\\u003eThree pairs of primers specific to the corresponding bacteria were used in the multiplex PCR assay (Table S1). The multiplex PCR was performed with reaction solution contained 2 \\u0026micro;L of each genomic DNA, 2 \\u0026micro;L of each primer (at a final concentration of 0.8 \\u0026micro;M), 25 \\u0026micro;L 2\\u0026times;Taq PCR Master Mix buffer and sterilized ddH\\u003csub\\u003e2\\u003c/sub\\u003eO to make final volume of 50 \\u0026micro;L. PCR reactions were carried out using a S1000TM thermal cycler (BioRad Laboratories, USA), the PCR condition included initial denaturation for 5 min at 95\\u0026deg;C, followed by 32 cycles of 95\\u0026deg;C for 30 s, 53\\u0026deg;C for 30 s and 72\\u0026deg;C for 30 s, and then a final extension at 72\\u0026deg;C for 10 min. PCR products were stored at -20\\u0026deg;C for further detection.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Conjugation of AuNPs with multiplex PCR products\\u003c/h2\\u003e \\u003cp\\u003eThe AuNPs was prepared by the Turkevich method (Kimling, 206) using sodium citrate as reducing agent with slightly modifications. Briefly, 100 \\u0026micro;L of 0.1 mM HAuCl\\u003csub\\u003e4\\u003c/sub\\u003e was added to 99.9 mL Milli Q water and stirred vigorously. After boiling, 1 mL of 1% sodium citrate was added quickly to the HAuCl\\u003csub\\u003e4\\u003c/sub\\u003e solution under stirring and boiling for another 10 min. The obtained red color solution will then cool to room temperature and stored at 4\\u0026deg;C in dark. The synthesized AuNPs were characterized by UV-vis spectrum, TEM and FT-IR analysis.\\u003c/p\\u003e \\u003cp\\u003ePCR products were conjugated with AuNPs in a acid environment, specifically, 5 \\u0026micro;L of 1 M citric acid buffer (PH 3) was added to 50 \\u0026micro;L PCR products, then 50 \\u0026micro;L AuNPs was transferred to the above solution and incubated for 3 min to make AuNP-PCR product conjugation. Afterward, the mixture was centrifuged at 4694 Xg for 5 min, the pellet was resuspended in 100 \\u0026micro;L of 10% BSA for blocking, then the AuNP-PCR product-BSA composite was obtained.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Fabrication of LFIA\\u003c/h2\\u003e \\u003cp\\u003eThe LFIA was prepared according to previous study (Wu, 2021), it consisted of four parts: sample pad (1.5 cm \\u0026times; 3 mm), NC membrane (2.5 cm \\u0026times; 3 mm), absorbent pad (3 cm \\u0026times; 3 mm) and backing pad (7 cm \\u0026times; 3 mm). Totally three test lines on the NC membrane was designed for the detection of the corresponding three strains. First test line (T1) was spotted with anti-biotin antibody, second test line (T2) was spotted with anti-FITC antibody and the third test line (T3) was spotted with anti-digoxin antibody at a rate of 1 \\u0026micro;L cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, respectively. Finally, the LFIA was dried at 37\\u0026deg;C for 4 h and then cut into 3 mm width, and further stored in a desiccator kept in dark.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e\\u003cem\\u003e2.6 Multiplex LFIA procedure for the detection of S.\\u003c/em\\u003e Typhimurium,\\u003cem\\u003eE. coli\\u003c/em\\u003e O157: H7 and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003eA series of concentrations of \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium,\\u003cem\\u003eE. coli\\u003c/em\\u003e O157: H7 and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e ranged from 10\\u003csup\\u003e9\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e to 10\\u003csup\\u003e1\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e were prepared to determine the limit of detection of the assay. After DNA extraction and PCR amplification, 20 \\u0026micro;L of the AuNP-PCR product-BSA suspension were detected by LFIA and results were observed in 10 minutes. As control, PCR products were analyzed using 2% agarose gel electrophoresis and observed under Gel Imager System (geldoc EZ, USA).\\u003c/p\\u003e \\u003cp\\u003eIn order to confirm the specificity of this assay, three common foodborne pathogens at a high concentration (10\\u003csup\\u003e7\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) were detected by the LFIA, including \\u003cem\\u003eS. aureus\\u003c/em\\u003e, \\u003cem\\u003eV. parahaemolyticus\\u003c/em\\u003e and \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e. All experiments were repeated three times to prove credibility.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7 Detection of artificially contaminated food samples\\u003c/h2\\u003e \\u003cp\\u003eChicken breast were obtained from a local supermarket and analyzed within 1 h after purchase. Samples were pretreatment according to the culture-based method. Briefly, chicken breast was divided into 3 portions of 25g and washed twice with deionized water, soaked in 75% ethanol for 10 min, and then sterilized with UV light for 30 min. Bacteria in varying concentrations were spread evenly each 25 g chicken breast and incubated overnight respectively. Then, the chicken breast was placed in 225 mL PBS, and treated with flapped and concussed to obtain the bacterial suspension and detected by the proposed method. At the same time, the plate count method was performed to verify the detection.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results And Discussion\",\"content\":\"\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec11\\\"\\u003e\\n \\u003ch2\\u003e3.1 The detection principle of multiplex LFIA\\u003c/h2\\u003e\\n \\u003cp\\u003eThe principle of the developed detection method of bacteria was demonstrated in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. AuNPs in red wine color was prepared by using sodium citrate as reducing and stabilizing agent. Bacterial DNA was extracted by boiling for 10 minutes, which is simple, rapid, economic and environmental friendly as compare with traditional solvent extraction and commercial kits. For multiplex PCR, three pairs of primers specific for \\u003cem\\u003ehut\\u003c/em\\u003e gene of \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, \\u003cem\\u003erfbE\\u003c/em\\u003e gene of \\u003cem\\u003eE. coli\\u003c/em\\u003e O157: H7 and \\u003cem\\u003ehly\\u003c/em\\u003e gene of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e were prepared. The forward primers for \\u003cem\\u003ehut\\u003c/em\\u003e, \\u003cem\\u003erfbE\\u003c/em\\u003e and \\u003cem\\u003ehly\\u003c/em\\u003e gene were labeled with SH group, and the reverse primers for \\u003cem\\u003ehut\\u003c/em\\u003e, \\u003cem\\u003erfbE\\u003c/em\\u003e and \\u003cem\\u003ehly\\u003c/em\\u003e gene were labeled with FITC, digoxin and biotin, respectively. As a result, multiplex PCR products contained both SH group and FITC amplified from \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium DNA, contained both SH group and digoxin amplified from \\u003cem\\u003eE. coli\\u003c/em\\u003e O157: H7 DNA, and contained both SH group and biotin and amplified from \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e DNA. The PCR products were then conjugated with AuNPs in citric acid buffer (pH 3), and the remaining sites on AuNPs were blocked by BSA. The AuNP-PCR product-BSA was detected by LFIA strip by adding it to sample pad and migrated along the LFIA toward absorbent pad. When it reached NC membrane, biotin would be captured by anti-biotin antibody on T1, FITC would be captured by anti-FITC antibody on T2, and digoxin would be captured by anti-digoxin antibody on T3. If samples contained target bacteria, the corresponding T lines would appear red color, which was attributed to the AuNPs. With the accumulation of PCR products, the red bands on the T line would be gradually deepened. On the contrary, no red band would appear on the T line if target bacteria were absent in the sample.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec12\\\"\\u003e\\n \\u003ch2\\u003e3.2 Optimization of multiplex PCR assay and antibody concentration on test line of LFIA\\u003c/h2\\u003e\\n \\u003cp\\u003eIn order to ensure the specific of the multiplex PCR and obtain each specific PCR product, annealing temperature (48.8℃, 50℃, 53℃, 55.5℃, 56.2℃, 58.8℃, 60℃, 62.2℃) and concentration of primer pairs (Table S2) were optimized in this study. The multiplex PCR products were 360 bp, 495 bp and 678 bp, corresponding to specific genes of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, respectively (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA and \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The gray value of the band brightness was measured and analyzed by image J software. It was found that the band brightness reached the maximum when the annealing temperature was 53℃ (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC) and the concentrations of the primer pairs was 0.8 \\u0026micro;M respectively (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). The antibody concentration on T line is critical for the detection and the cost of LFIA. The anti-biotin antibody, anti-FITC antibody and anti-digoxin antibody at the concentration of 0.4M, 0.6M, 0.8M and 1M were optimized respectively. It was found that red bands reached the maximum when the concentrations of all three antibodies were 1M (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). The intensity of red bands analyzed by image J software were further confirmed the optimal concentration of all three antibodies is 1M (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE insert image). Therefore, the annealing temperature at 53℃ and the concentrations of all three pairs of primers at 0.8 \\u0026micro;M, and all three antibodies at concentration of 1M were used as optimal conditions for the detection.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec13\\\"\\u003e\\n \\u003ch2\\u003e3.3 Characterization of AuNPs and AuNP@PCR products\\u003c/h2\\u003e\\n \\u003cp\\u003eThe AuNPs were synthesized by HAuCl\\u003csub\\u003e4\\u003c/sub\\u003e and sodium citrate through heating stirring process. As demonstrated in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, the AuNPs were uniformly elliptical spherical and the size were approximately 50\\u0026ndash;75 nm, with maximum absorption peak at 525 nm.. The size of AuNP@PCR products were increased to approximately 100\\u0026ndash;150 nm,. Furthermore, the zeta potential of AuNPs and AuNP@PCR products were \\u0026minus;\\u0026thinsp;26.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.2 mV and \\u0026minus;\\u0026thinsp;37.75\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5 mV, respectively (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF), indicating the AuNPs was successfully conjugated with PCR products. According to the FTIR spectra of AuNPs, peaks at 1249, 1452 and 3240 were due to C-CH3 tensile vibration and the bending vibration of methylene C-H, O-H stretching vibration of hydroxyl functional groups, respectively (Sivakumar, 2009; Sharma, 2012; Ganapuram, 2015; Tanzil, 2016; Babu. 2011; Babu, 2012), the FTIR spectra of AuNP@PCR products showed more peaks than AuNPs, due to the existent of DNA, biotin, FITC digoxin, etc, its functional groups are more complex.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec14\\\"\\u003e\\n \\u003ch2\\u003e3.4 Sensitivity of the assay\\u003c/h2\\u003e\\n \\u003cp\\u003eThe limit of detection of genomic DNA by the LFIA strip was firstly studied. Serial dilutions (70, 35, 17.5, 8.8, 4.4, 2.2, 1.1, 0.6, 0.3, 0.15 and 0.075 ng/\\u0026micro;L) of each bacteria\\u0026apos;s DNA was prepared. The DNA were was subjected to multiplex PCR assay, and sterilized ddH\\u003csub\\u003e2\\u003c/sub\\u003eO was performed as control in the same manner. After multiplex PCR amplification, PCR products were conjugated with AuNPs and then detected by LFIA. The PCR products were analyzed using 2% agarose gel electrophoresis and observed under Gel Imager System (geldoc EZ, USA) as control. The limit of detection of genomic DNA were found to be 150 pg/\\u0026micro;L for \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e,150 pg/\\u0026micro;L for \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, and 600 pg/\\u0026micro;L for \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA and \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). The limit of detection of DNA examined by traditional agarose gel method were 1.1 ng/\\u0026micro;L for \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 (Fig.S1A), which is less sensitive than the LFIA proposed by this study.\\u003c/p\\u003e\\n \\u003cp\\u003eThe detection sensitive for the three bacteria of the developed method was studied under the optimized conditions. Different concentrations of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 (10\\u003csup\\u003e1\\u003c/sup\\u003e -10\\u003csup\\u003e9\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) were prepared by diluting the logarithmic phase bacteria with sterile PBS, and quantified by plate count method. Results showed that the limit of detection of multiplex PCR products for \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 by traditional agarose gel were 0.9\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e, 1.2\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e, 1.5\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, respectively (Fig.S1B). As demonstrated in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC, the detection limit of \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e were 1.6\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1.0\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1.0\\u0026times;10\\u003csup\\u003e1\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e by the LFIA with naked eye. The detection sensitivity of the LFIA proposed in this study was improved 10\\u003csup\\u003e6\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e for \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e and 10\\u003csup\\u003e5\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e for \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, as compared with traditional multiplex PCR assay. This improvement is due to AuNPs signal amplification and LFIA convenient and fast detection, whereas agarose gel is time-consuming, toxic, and requires gel imager equipment. Moreover, according to the analysis by Image J, intensity of T lines has a good linear relationship with the concentration of corresponding bacteria. The linear equation of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e concentration in the range of 1.0\\u0026times;10\\u003csup\\u003e1\\u003c/sup\\u003e to 0.8\\u0026times;10\\u003csup\\u003e9\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e was Y\\u0026thinsp;=\\u0026thinsp;33.29\\u0026thinsp;+\\u0026thinsp;32.50 X with correlation coefficient (R\\u003csup\\u003e2\\u003c/sup\\u003e) of 0.941 (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD), \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium concentration in the range of 1.0\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e to 1.1\\u0026times;10\\u003csup\\u003e9\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e was Y= -74.08\\u0026thinsp;+\\u0026thinsp;57.02 X with correlation coefficient (R\\u003csup\\u003e2\\u003c/sup\\u003e) of 0.996 (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE), \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 concentration in the range of 1.6\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e to 1.5\\u0026times;10\\u003csup\\u003e9\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e was Y= -64.36\\u0026thinsp;+\\u0026thinsp;43.91X with correlation coefficient (R\\u003csup\\u003e2\\u003c/sup\\u003e) of 0.962 (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF).\\u003c/p\\u003e\\n \\u003cp\\u003eThe multiplex LFIA employed PCR amplification and AuNPs labeling signal to improve the sensitivity of the detection of foodborne pathogenic bacteria. By comparing with some recently reported PCR-based methods and AuNP-based methods for detection of foodborne pathogenic bacteria, the proposed method showed better performance in terms of sensitivity and rapid (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The developed multiplex strips are faster, more sensitive and less time consuming than single T line strip when detecting of three species of bacteria. \\u0026nbsp;\\u003c/p\\u003e\\u0026nbsp;\\u003ctable border=\\\"1\\\" id=\\\"Tab2\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eComparison of reported multiplex detection methods for target bacteria\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eDetection method\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTarget\\u003c/p\\u003e\\n \\u003cp\\u003ebacteria\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eDetection limit\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTotal time\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eReference\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAntibody-AuNP-MNPs\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" rowspan=\\\"5\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eSA\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eSE, EC\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eEC, YE, ST, LM\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eLM, ST, EC\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eLM, ST, EC\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eLM, ST, EC\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.5\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003eCFU/mL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026gt;\\u0026thinsp;24 h\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e(Sung,2013)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7-plex PCR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3 CFU/10g\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026gt;\\u0026thinsp;16 h\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e(Wang, \\u003cspan class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eChemiluminescence-IMS\\u003c/p\\u003e\\n \\u003cp\\u003ereal-time PCR-SEL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003csup\\u003e4\\u003c/sup\\u003e CFU/mL\\u003c/p\\u003e\\n \\u003cp\\u003e10 CFU\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026gt;\\u0026thinsp;18 h\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026gt;\\u0026thinsp;21 h\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e(Magliulo, 2007)\\u003c/p\\u003e\\n \\u003cp\\u003e(Wang, \\u003cspan class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eIMS-PMA-mPCR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003csup\\u003e2\\u003c/sup\\u003e CFU/mL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.5 h\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e(Yang, 2013)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003emPCR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003csup\\u003e2\\u003c/sup\\u003e CFU/mL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4 h\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThis work\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003cp\\u003e\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec15\\\"\\u003e\\n \\u003ch2\\u003e3.5 Specificity of the assay\\u003c/h2\\u003e\\n \\u003cp\\u003eThe multiplex LFIA was tested for the specificity against three other foodborne pathogen bacteria including \\u003cem\\u003eS. aureus\\u003c/em\\u003e, \\u003cem\\u003eV. parahaemolyticus\\u003c/em\\u003e and \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e. The traditional agarose gel verified the accurate and specific of muplex PCR (Fig.S1C). As demonstrated in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and B, the appearance of red band on the separate T line was only observed when corresponding target bacteria is present. No significant changes were observed in the presence of other disturbing foodborne pathogen bacteria. Therefore, the multiplex LFIA is highly specific and ensures accurate detection of target bacteria.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec16\\\"\\u003e\\n \\u003ch2\\u003e3.6 Detection of bacteria in food samples\\u003c/h2\\u003e\\n \\u003cp\\u003eThe prepared chicken breast samples spiked with \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 (10\\u003csup\\u003e1\\u003c/sup\\u003e-10\\u003csup\\u003e8\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e), \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium (10\\u003csup\\u003e1\\u003c/sup\\u003e-10\\u003csup\\u003e8\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) and \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e (10\\u003csup\\u003e1\\u003c/sup\\u003e-10\\u003csup\\u003e8\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) were tested by the multiplex LFIA strip, the recovery rates and their coefficients of variation were calculated, and the accuracy of the proposed method was evaluated by plate count method. Traditional agarose gel method was also used to detect the multiplex PCR products as control, and the limit of detection turned out to be 0.5\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e, 1.9\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e, 3.7\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, respectively (Fig.S1D). For comparison, a commercial \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e LFIA strip, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium LFIA strip and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 LFIA (Meizheng Bio, China) were employed to detect \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, respectively. The limit of detection were 9.2\\u0026times;10\\u003csup\\u003e7\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1.2 \\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1.6 \\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, respectively (Fig.S2). Therefore, the developed multiplex LFIA assay in this study is more sensitive and more efficient as compare with commercial kit.\\u003c/p\\u003e\\n \\u003cp\\u003eAs demonstrated in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC, the limit of detection of the developed multiplex LFIA strip of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 was 0.3\\u0026times;10\\u003csup\\u003e1\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 2.0\\u0026times;10\\u003csup\\u003e1\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e and 3.5\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, respectively. As compare with commercial LFIA strips, the limit of detection of developed multiplex LFIA strip was improved 10\\u003csup\\u003e6\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003eof \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, 10\\u003csup\\u003e5\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003eof \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and 10\\u003csup\\u003e4\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003eof \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7, and the result can be read in one strip, which is convenient and low cost. The intensity of red band on T lines were gradually increased with the increasing of bacteria concentration (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD-F). The recoveries of this study were ranging from 92.7\\u0026ndash;112.1%, with the coefficient of variation less than 8.73%, and the recoveries of plate count were in the range of 96.6\\u0026ndash;102.9% with the coefficient of variation less than 7.28% (Table \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Therefore, the developed multiplex LFIA is sensitive and accurate, and shows a promising application in simultaneous detection of foodborne pathogen bacteria in food samples.\\u0026nbsp;\\u003c/p\\u003e\\u0026nbsp;\\u003ctable border=\\\"1\\\" id=\\\"Tab3\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eAnalytical results of bacteria spiked in chicken breast by this detection system and using plate counting (n\\u0026thinsp;=\\u0026thinsp;3).\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eSpiked\\u003c/p\\u003e\\n \\u003cp\\u003e(lgCFU/mL)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eMean found\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD (lgCFU/mL)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eRecovery (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eCV (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThis method\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePlate counting\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThis method\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePlate counting\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThis method\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePlate counting\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eLM\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.71\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.07\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e99.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e102.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.23\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e100.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e98.0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.36\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.73\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.15\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e106.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e97.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.90\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eST\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e105.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e96.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.17\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.29\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.82\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.66\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e103.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e99.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.29\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.55\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e112.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e102.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8.73\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1.84\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\" rowspan=\\\"3\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eEC\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.58\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.55\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.13\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.97\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e92.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e99.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3.81\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2.44\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.58\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.55\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.26\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e92.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e100.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.23\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.47\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.58\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7.57\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e104.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e97.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4.69\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.60\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003cp\\u003e\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eIn the present study, a novel multiplex LFIA was developed for simultaneous detection of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 by naked eye. The highly sensitive of this method was take advantages of PCR amplification and AuNPs labeling. And benefit by the specific of primer and antibody, this method was highly reliable. The detection limitation of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 was 1.0\\u0026times;10\\u003csup\\u003e1\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1.0\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e and 1.6\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, respectively. The assay took 4 h including 1.5 h for multiplex PCR and 2 h for preparing AuNP@PCR product@BSA, and finally LFIA strip detection. Thus, the developed method was fast, sensitive, specific and reliable for the detection of the three target bacteria in food sample, and could be applied for simultaneous detection of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium, \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eAcknowledgments: This work was funded by the major special project on public welfare of Henan Province [grant number 201300110100].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompliance with Ethical:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis manuscript has not been published or presented elsewhere in part or in entirety,\\u0026nbsp;and is not under consideration by another journal.\\u003c/p\\u003e\\n\\u003cp\\u003eFunding: This work was funded by the major special project on public welfare of Henan Province [grant number 201300110100].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e: The authors declared that they have no conflicts of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e: Dianbo Zhao: Conception, Methodology, Writing-review. Jialei Liu: Material preparation, Data collection, Writing-original draft. Juan Du: Analysis and Writing-editing. Kai Liu: Material preparation. Yanhong Bai: Funding acquisition, Resources, Supervision.\\u0026nbsp;All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval\\u003c/strong\\u003e: This article does not contain any studies with human participants or animals performed by any of the authors.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInformed consent:\\u003c/strong\\u003e Informed consent was obtained from all individual participants included in the study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eD\\u003c/strong\\u003e\\u003cstrong\\u003eata availability statemen\\u003c/strong\\u003e\\u003cstrong\\u003et\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eBabu PJ, Sharma P, Kalita MC. 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Solvothermal synthesis of \\u0026alpha;-Fe\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e3\\u003c/sub\\u003e polyhedrons and its application in an immunochromatographic strip test for the detection of foodborne pathogen \\u003cem\\u003eListeria monocytogenes. \\u003c/em\\u003eNanotechnology,\\u003cem\\u003e \\u003c/em\\u003e32(2021), 085502., \\u003cu\\u003ehttps://doi.org/10.1088/1361-6528/abcb30.\\u003c/u\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003eYang X, Bai XN, Yang JYJ, Xu F, Xu HY. (2013). Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella\\u003cem\\u003e Typhimurium\\u003c/em\\u003e, \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e O157:H7 and \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e in food products. Food Microbiology, 34(2), 418-424. \\u003cu\\u003ehttps://doi:10.1016/j.fm.2013.01.004.\\u003c/u\\u003e \\u003c/li\\u003e\\n\\u003cli\\u003eYang X, Bai XN, Zhang J. (2020). Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China. International Journal of Medical Microbiology, 310(1), 151377. https://doi:10.1016/j.ijmm.2019.151377.\\u003c/li\\u003e\\n\\u003cli\\u003eYang X, Zhou X, Zhu M. (2017). Sensitive detection of \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e based on highly efficient enrichment with vancomycin-conjugated brush-like magnetic nano-platforms. Biosensors \\u0026amp; Bioelectronics, 91(2017), 238\\u0026ndash;245. \\u003cstrong\\u003e\\u003cu\\u003ehttps://doi:10.1016/j.bios.2016.11.044.\\u003c/u\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cu\\u003e \\u003c/u\\u003e\\u003c/strong\\u003e\\u003c/li\\u003e\\n\\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\":\"info@researchsquare.com\",\"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\":\"multiplex lateral flow immunoassay, multiplex PCR, gold nanoparticles, foodborne pathogenic bacteria\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2073305/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2073305/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eIn this study, a sensitive, fast and reliable multiplex lateral flow immunoassay based on multiple PCR and gold nanoparticles (AuNPs) was developed. Genomic DNA of \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eSalmonella\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e O157:H7 was extracted by a simple boiling method. Three pairs of primers were designed and labeled according to specific gene fragment of the three strains for multiple PCR. The PCR products were then conjugated with AuNPs and detected by multiplex lateral flow strip, on which the test lines loaded with anti-biotin antibody, anti-FITC antibody and anti-digoxin antibody corresponding to the labels of primers, respectively. Results showed the limit of detection of \\u003cem\\u003eL. monocytogenes\\u003c/em\\u003e, \\u003cem\\u003eS.\\u003c/em\\u003e Typhimurium and \\u003cem\\u003eE. coli\\u003c/em\\u003e O157:H7 in pure culture were 1.0\\u0026times;10\\u003csup\\u003e1\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1.0\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e and 1.6\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, respectively, without culture enrichment. In addition, the lateral flow immunoassay showed good specificity, no reaction to each other or no cross-reactivity with other tested foodborne bacteria were observed. The detection took less than 4 h including PCR amplification, AuNPs conjugation and strip detection. Furthermore, the developed method was applied for the detection of food samples (chicken breast), which was verified by plate count method. The recoveries ranged from 92.7\\u0026ndash;112.1%, with the coefficient of variation less than 8.73%, revealing the feasible and reliable application of this method in practical sample. Therefore, the developed multiplex lateral flow strip is sensitive, accurate and visualized, which is applicable to simultaneous detection of the three foodborne pathogenic bacteria in food sample.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A highly sensitive multiplex lateral flow immunoassay for simultaneous detection of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2022-09-27 13:57:56\",\"doi\":\"10.21203/rs.3.rs-2073305/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"9726d84c-b7b8-44f9-8f3f-5ee0d4d6a1ee\",\"owner\":[],\"postedDate\":\"September 27th, 2022\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-01-16T01:29:16+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2022-09-27 13:57:56\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2073305\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2073305\",\"identity\":\"rs-2073305\",\"version\":[\"v1\"]},\"buildId\":\"-D5TCW68w8eVRRLyjaTIo\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}