| The study aims to develop a multiplex detection method for Salmonella(Sa), Vibrio parahaemolyticus(Vp), and Vibrio cholerae(Vc), including a multipathogen selective enrichment broth and an Fluorescence Quantitative multi-PCR (FQ-mPCR). The detection method can inspect the main pathogens in seafood through one-step enrichment and one-step detection. It can greatly shorten the time needed, simplify the detection procedure and improve the detection efficiency.BPW was selected as an initial medium. The additives which could improve the growth of target pathogens or inhibit that of non-target pathogens were selected to conduct single factor experiments and response surface methodology in BPW. The optimal formula of the SVV broth was as follow: BPW 20.1g/L, NaCl 20g/L,potassium tellurit 1mg/L,bile salt no.3 2.5g/L,sodium citrate 5g/L,glucose 1.25g/L,mannitol 1.25g/L,anhydrous sodium sulfite 1.25g/L and sodium pyruvate 0.05g/L. The results showed that when grow independently, or mixed, the target pathogens had a great accumulation (105-108 cfu/mL) after incubated in SVV for 18h at 37℃with shaking. It could also inhibit the growth of competitive microflora effectively. At the same time, the SVV broth had the extensive applicability. The SVV enrichment combine with PCR detection could inspect the pathogens effectively and the accuracy is 100%.We desingned several pairs of primers and TaqMan fluorescent probes targeting the special sequences of conservative invA gene of Sa, toxR gene of Vp, and hlyA gene of Vc. The most efficient primers and probes were found by screening their combinations. BSA 0.04%(w/v) and formamide 0.01%(w/v) was used to enhance the anti-interference ability of the assay. The FQ-mPCR was developed by optimizing the amplification system and conditions. The 25μL amplification system contains:1×Buffer,3.75mmol/L MgCl2, 1mmol/L dNTP mixture,0.5mmol/L upper and lower primers of Sa, 0.4mmol/L of Vp, 0.46mmol/L of Vc, and 0.18μmol/L probe of of Sa,0.2μmol/L of Vp,0.22μmol/L of Vc,2.5U Taq DNA polymerase, 2μL DNA template; amplification procedure:94℃4min,94℃10S, 60℃45S,40cycle. The specificity of the PCR assay is 100%. The sensitivity of the PCR for detection of target pathogens was 1300cfu/mL for Sa,6400 cfu/mL for Vp,2100 cfu/mL for Vc.52 clinical samples were detected using SVV with FQ-mPCR. The results show that it can effectively prevent undetection and the accuracy is 100%.The developed multipathogen selective enrichment broth SW broke through the traditional two step enrichmen method. It could afford the simultaneous growth of target pathogens and effectively improve the sensitivity and accuracy of the following FQ-PCR assay. The developed FQ-mPCR conbines with the simple DNA extraction method could greatly simplify the detection procedure and ensure the pathogens can be detected. In conclusion, the developed multiplex detection system--multipathogen selective enrichment broth conbine with FQ-mPCR detection could quickly and effectively inspect Sa, Vp, and Vc, and thus could be used in entry-exit inspection and quarantine of seafood, as well as in daily inspection of other food. |
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