Detecing The Enterotoxin Of ETEC Using SYBR Green I Q-PCR

Diarrhoea of young pig was one of the important diseases that affect the healthy development of the pig breeding. Although the causes of diarrhoea in pigs were complex, the Enterotoxigenic Escherichia coli (Enterotoxigenic E.coli, ETEC) was recognised as the most frequent causes of piglet diarrhoea. At present, many scholars at home or abroad studied the diarrhea pathogenesis, diagnosis and prevention of ETEC, and had made great progress, but the proliferation of ETEC from pig in the small intestine was less limited. According to the theory of SYBR Green dye, this research establishes the method of real time quantitative PCR to detect the LT and STa gene, which was specially and rapidly detected ETEC from porcine. Furthermore, this study also used the Q-PCR method to detect the proliferation of ETEC from porcine in small intestine of mice, in order to provide experimental data to further elucidate the vivo pathogenesis of ETEC. Results are as follows:1. The amplified products were obtained by conventional PCR, which was 314bp length, then the nucleotide sequences were cloned, the recombinant plasmid was tested by PCR and sequenced.According to the result of sequencing, a pair of fluorescent primers was designed, the plasmids were diluted as standard. The standard curve of LT was Y=-3.33X+42.77, using SYBR GreenⅠreal-time quantitative PCR, the related coefficient was 0.999, the amplification efficiency was 99.7%, the sensitivity is 4.09×103 copies/μL. Specific results showed that E.coli C83912 (STa+) and ATCC25922 were negative, while C83903 (LT+) and the recombinant plasmid pMD19-LT was positive, which indicated the designed primer is special for LT gene. Repeatability results showed inter and intra coefficient of variation of repeatability test were less than 3%, indicating that the experimental data was good stability and repeatability.2. According to published sequences of STa in GeneBank (accession number M25607), the primers were designed by conventional PCR and obtained the amplified fragment which was 323bp length.The recombinant plasmid was tested by PCR and sequenced.According to the result of sequencing, a pair of fluorescent primers was designed, the plasmids were diluted as standard. The standard curve of STa was Y=-3.271+47.902, using SYBR GreenⅠreal-time quantitative PCR, the related coefficient was 0.999, the amplification efficiency was 99.7%. The sensitivity reached 8.85×10~2 copies/μL, specific test results showed E. coli C83903 (LT +) and ATCC25922 were negative, while C83912 (STa +) and the recombinant plasmid pMD19-STa positive, indicating the primer was can only amplify the fragments of STa. The results showed that with 108,106,103 repeatability coefficient of variation were 1.08%, 0.92% and 0.61% and between batch coefficients of variation were 1.22%, 1.86% and 1.51% which were less than 2%, indicating that the method has good accuracy and repeatability.3. Using the Escherichia.coil C83903 (LT+) and C83912 (STa+) infected mice, the diarrhea models were builded. The contents of small intestinal from mice infected was collected, and deteted by the real-time PCR. The results are as follows: the first 4h, 16h and 2d samples of infected mice with pathogenic ETEC (LT+) were positive, while the first 7d and 14d samples were negative; the first 4h, 16h, 2d, 7d and 14d samples of infected mice with pathogenic ETEC (STa+) were all positive.These results suggested that the proliferation of LT and STa enterotoxin was different in infecting intestinal tissueed and provided further understanding of pathogenesis of ETEC infection in animals from the molecular level.