| ObjectivePCR reaction conditions of 14 kinds of foodborne pathogens were optimized and 14 kinds of foodborne pathogens were divided into two groups according to the annealing temperature and the size of amplification products.14 kinds of foodborne pathogens could be amplificated in two PCR reactions through optimization of PCR reaction conditions divided into two groups, thus test time of pathogens was shortened and this will win time for handling epidemic situation. Finally, a rapid, sensitive, specific pathogens detection method was established, which provided timely and accurate information for handling the incident scene and treatment of patient.MethodsA part of pathogens primers were obtained from references, and another part of primers were designed using Primer 5.0 software according to pathogens specific gene sequences from GenBank database.14 kinds of foodborne pathogens were divided into two groups according to the annealing temperature and the size of amplification products. Then, multiple PCR and many multiple PCR reactions were optimized.ResultsThe paper gained many results through studying fourteen pathogens detection using multiplex PCR and united multiplex PCR, as follows.1. Fourteen pathogens were divided intothree groups according to annealing temperature and amplification products sizein order to amplify them one time through united multiplex PCR. Grouping scheme are as follows, the first scheme: (1) Salmonella Enteritidis, Vibrio parahaemolyticus, Enterobacter sakazakii, Proteus mirabilis and Campylobacter jejuni, (2) Shigella flexneri, Enterohemorrhagic E. coli O157:H7, Staphylococcus aureus, Listeria monocytogenes, Vibrio vulnificus and Bacillus thuringiensis, (3) Bacillus cereus, Streptococcus hemolyticus and Yersinia enterocolitica; the second scheme: (1)Salmonella Enteritidis, Shigella flexneri, Vibrio parahaemolyticus, Vibrio vulnificus and Campylobacter jejuni, (2) Enterohemorrhagic E. coli O157:H7, Staphylococcus aureus, Bacillus thuringiensis, Enterobacter sakazakii and Proteus mirabilis, (3) Listeria monocytogenes, Bacillus cereus, Streptococcus hemolyticus and Yersinia enterocolitica.2. Annealing temperature, primers amount, sensitivity and primers proportion of multiplex PCR and united multiplex PCR were optimized. The optimum annealing temperature of united multiplex PCR was 52℃; The optimum mixed primer amount of united multiplex PCR was 0.6-0.8μL; Genomic DNA detection sensitivity of united multiplex PCR was up to 8×10-4 ng/μL; Primers proportion of the first scheme were:1:1:3:3:2,1:1:1:1:3:3 and 1:1:2, respectively; Primers proportion of the second scheme were:1:1:1:3:2,1:1:3:3:2 and 1:1:1:2, respectively.3. Vibrio parahaemolyticus was detected successfully from patients’vomit, stool and food (had eated by patients) samples using united multiplex PCR optimized.4. The study compared annealing sensitivity and application effect in detection food poisoning samples of common PCR with multiplex PCR. The results indicated that Sensitivity of common PCR was up to 2×10-7 ng/μL, and Sensitivity of multiplex PCR was up to 2×10-6 ng/μL. It was easier to detect pathogens through common PCR than PCRmultiplex PCR.ConclusionThe fourteen pathogens could be detected completely in the same batch using united multiplex PCR, and the study had established a rapid, simple, accurate, sensitive, specific and effective pathogen detection method. These results had provided accurate information for field investigation and settlement and treatment patients’ inemergency. |
