Establishment and Application of Multiplex Integrated qPCR System for 16 Kinds of Pathogenic Microorganisms in Raw Milk
In order to meet the need of rapidly detecting microorganisms in raw milk at a high throughput,a multiplex integrated real-time quantitative PCR(qPCR)was established to rapidly detect 16 pathogenic microorganisms that might be available in raw milk,specific primers and probes for the target strains including pathogenic Escherichia coli,Staphylococcus aureus,Streptococcus spp.and other bacteria were designed,after optimizing the reaction system and evaluating the specificity and sensitivity of the method,a stable 4-group multiplex qPCR system was established,and compared with dot immunogold filtration assay(DIGFA)using artificially contaminated raw milk samples.The results showed that all the primers and probes could effectively amplify targeted strains,without cross-reaction in each group or specific reaction to 12 non-targeted strains in other 3 groups;the coefficients of variation(CVs)of the intra-and inter-groups were both lower than 3%;its lowest detection limit was 102 CFU/mL for Brucella,103 CFU/mL for 8 pathogenic microorganisms such as Enterobacter sakazakii,Bacillus cereus and Salmonella,and 104 CFU/mL for 7 pathogenic microorganisms such as Shigella,Campylobacter and Streptococcus;for each of 25 samples artificially contaminated with pathogenic bacteria,the test results by the established method were basically consistent with those by DIGFA.In conclusion,the established multiplex integrated qPCR system,with good sensitivity,specificity and reproducibility,could be used for rapid and efficient detection of various pathogenic microorganisms in raw milk samples,supporting the microbiological risk monitoring of raw milk.
万方数据
维普
