| Nowadays, foodborne diseases coursed by pathogenic microorganisms still present a tough issue to food security and public health in the world, such as Vibrio cholerae, Vibrio parahemolyticus and Salmonella spp., Vibrio parahemolyticus and Salmonella spp. are two common pathogenic bacteria causing food poisoning of human, while Vibrio cholerae is responsible for a virulent intestinal infectious disease called Cholera which is classified into A class infectious diseases list of China. Therefore, the establishment of rapid and effective detection approaches is crucial. Recently, techniques like polymerase chain reaction(PCR), real-time PCR(q PCR), Enzyme-linked immunosorbent assay(ELISA), Gene chip technology for detecting pathogens have developed rapidly and maturely. However, these assays have several drawbacks. Professionals were needed in some steps and reaction reagents used were expensive that rural areas can not afford. In addition, all of rapid testing methods are incapable to distinguish viable cells from dead cells virtually which may enhance the possibility of positive results and overestimate the enumeration of bacteria and the risk of food security. To solve problems mentioned above, here a real-time visual PMA-LAMP assay was developed in this study for detecting these three foodborne pathogens. On one hand, this assay estimated have handled two problems of methodology of the LAMP assay. And on the other hand, we provided a new idea and technical support to diagnosis of food safety.Our study designed primers of LAMP and q PCR targeting thy A gene(Gen Bank Accession No.AY143429.1) of Vibrio cholerae, tox R gene(Gen Bank Accession No.GQ228073.1) of Vibrio parahemolyticus, inv A gene(Gen Bank Accession No.M90846.1) of Salmonella spp. and fim Y gene(Gen Bank Accession JQ665438.1) of Salmonella spp.. After a serial of optimization trials, a total of 25 μL visual LAMP reaction system was determined as follow: 20 m M Tris-HCl(p H 8.8), 10 m M KCl, 8 m M Mg SO4, 10 m M(NH4)2SO4, 0.1% Tween 20, 0.8 M Betaine, 1.4 m M each of d NTPs, 0.4 μM F3 and B3, 3.2 μM FIP and BIP, 1.6 μM LF and LB, 0.3 M Calcein, 0.5 M Mn Cl2, 8U Bst DNA polymerase, 2.5 μL DNA templates. The real-time visul LAMP was performed at 65°C for 60 min and the entire amplification was accomplished in the real-time PCR equipment. The reaction result of LAMP was able to by deserved by naked eyes through a change of Calcein in color. And at the same time, details of reaction could be monitored by fluorescent curves generated with q PCR analysis software.The specificity experiments showed that primers of LAMP and q PCR designed for Vibrio cholerae, Vibrio parahemolyticus and Salmonella were identified as high specificity and only strains related could by detected. To determine the sensitivity of the assay, ten-fold serial dilutions of Vibrio cholerae, Vibrio parahemolyticus and Salmonella spp. pure culture bacteria were used. Detection limits of Vibrio cholerae, Vibrio parahemolyticus and Salmonella with the assay were 1.1×102 CFU/m L, 5.0×101 CFU/m L and 6.3×102-6.3×103 CFU/m L, respectively. Results were in accordance with q PCR assay.Reaction conditions of PMA were optimized as follow. The incubation time in darkness was 5 min while the exposure time under LEDs was 30 min. Treating concentrations of PMA were 15 μM, 20 μM and 5 μM for Vibrio cholerae, Vibrio parahemolyticus and Salmonella, respectively. The detection limits of real-time visual PMA-LAMP assay and PMA-q PCR assay for detecting viable cells of three bacteria were both 1.1×102 CFU/m L, 5.0×102 CFU/m L and 6.3×103 CFU/m L. In artificial contaminated experiments, the result showed that real-time PMA-LAMP assay could detect 1.7×102 CFU/m L of Vibrio cholerae, 1.9×102 CFU/m L of Vibrio parahemolyticus and 6.3×103 CFU/m L of Salmonella.In the practical sample detection, we compared the real-time visual PMA-LAMP assay with PMA-q PCR assay and conventional culture method. Unknown samples purchased from local markets such as seafood, vegetables and eggs were pre-enriched prior and then detected by visual three methods mentioned above. In 40 pieces of samples, the positive detection rate of real-time visual PMA-LAMP assay for Vibrio cholerae was 2.5%. In 72 pieces of samples, the positive detection rate of real-time visual PMA-LAMP assay for Vibrio parahemolyticus was 23.6%. And the positive detection rate of real-time visual PMA-LAMP assay for Salmonella was 0%. The detection time of real-time visual PMA-LAMP assay was shorter than conventional culture time and the operation of the assay was more simple than PMA-q PCR.In this study, we combined the real-time visual LAMP method with PMA dye and developed a rapid PMA-LAMP assay for detecting Vibrio cholera, Vibrio parahemolyticus and Salmonella. This assay not only has many advantages of LAMP, but also keeps the characteristic of PMA. Besides, we also take some preliminaries for semi real-time detection of LAMP. In conclusion, the real-time visual PMA-LAMP assay estimated in the study can provide more technical reference for testing viable food microorganisms, especially in rural and remote sites. |
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