Development Of A Multiplex Pcr Method For Detection Of Enterotoxigenic Escherichia Coli In Fresh Pork

Enterotoxigenic Escherichia coli(ETEC): an important foodborne pathogen, can cause diarrhea, enteritis, cholera syndrome, severe dehydration and even death in humans, which has important public health significance. ETEC have the ability to colonize the small intestine, and no damage or intrusion effect to intestinal mucosa epithelial cells. ETEC usually produce the heat-labile(LT) and/or heat-stable(ST) enterotoxins that initiate secretory diarrhea. One of the major causes of ETEC infections in humans is the ingestion of food or water contaminated with these organisms. The traditional methods for ETEC detection in food are time and effort-consuming. However, PCR assays for detection of ETEC have the advantages of rapid and accurate. Thereinto, multiplex PCR technology has been widely applied for its high specificity, efficiency and sensitivity. Primers targeting the lt and sta virulence genes were designed in this study, and we developed a multiplex PCR method for detection of ETEC in fresh pork. The main conclusions were as follows:(1) We have developed a multiplex PCR assay with three pairs of primers, which can identify 2 pathogenic genes of ETEC simultaneously, and the uidA gene was applied as an internal standard for ETEC identification.(2) In order to develop an efficient multiplex PCR, reaction conditions were optimized. PCR conditions in a thermal cycler were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 60.2°C for 40 s, 72°C for 1 min, then followed by a 10-min extension step at 72°C. The multiplex PCR reaction system(25 μl): 12.5μL 2×Taq MasterMix, 0.8 μl of each forward and reverse primer at 10 μmol/L and 7.7μL of sterile ultrapure water, DNA for amplification was released from the single colony. The optimized multiplex PCR system can distinguished between ETEC and other control bacterias. Furthermore, the specificity of primers and this system were confirmed by PCR assay, the primers generated the expected specific respectively, and there was no nonspecific amplification phenomenon with other non-target bacterias.(3) A total of 30 fresh pork samples were collected from local markets, and ETEC was not detected by using multiplex PCR. But as few as 4 CFU/10 g of artificially contaminated pork sample could be detected,and samples without the inoculation of target ETEC cells showed negative results.(4) Another characteristic of this multiplex PCR assay is that it was carried out by colony PCR, giving it a higher sensitivity than the method using extracted genomic DNA as template. And there were no food inhibitory factors in our colony PCR. With the procedure of extracting DNA has been omitted and the Premix Taq was used in the PCR system, what makes this multiplex PCR assay easier to perform and requires lower levels of laboratory equipments.This method can be used to identify these organisms in fresh pork samples in food testing laboratories, so that the ETEC-positive samples can be quickly identified and proper actions taken to prevent illness.