Establishment And Primary Application Of Single And Multiplex QPCR Methods For Detection Of Five Virulence Factors Of Porcine E.coli

Among many piglet diarrheal diseases,bacterial diarrheal diseases are particularly common in pig farms.Piglet colibacillosis caused by pathogenic Escherichia coli has a greater impact on suckling piglets,causing colibacillosis in piglets characterized by acute diarrhea,dehydration and weight loss.The disease has the characteristics of strong contagiousness,multiple transmission routes and large scope of influence.At present,its treatment methods are limited,which seriously endangers the growth and development of piglets,and has become a diarrheal disease of widespread concern in pig farms.Piglets of different ages infected with different serotypes of E.coli will show different symptoms.Currently,piglet colibacillosis mainly refers to piglet yellow diarrhea,piglet pullorum,weaned piglet diarrhea and weaned piglet edema disease.At present,conventional biological differential diagnosis methods for bacterial diseases are time-consuming and labor-intensive,with low sensitivity and low specificity.Therefore,it is particularly important to establish fast and efficient detection methods for bacterial pathogenic factors to accurately diagnose pathogenic E.coli,so as to achieve early detection and prevention of diseases.In this study,five single-plex fluorescent quantitative PCR detection methods were first established to detect F4,F5,F6 fimbriae genes,LT genes,and Stx2e genes of E.coli.Found the conserved regions of genes on NCBI,design five pairs of specific primers and Taq Man probes,then optimized the annealing temperature and primer probe concentration,established a standard curve,and evaluated the sensitivity,specificity and repeatability of each method.The results showed that the standard curve correlation coefficient R~2of the five methods were all greater than 0.990,and the linear relationship was good.The minimum detection amount of F4,F5,F6 fimbriae and LT was 10~1copies/μL,and the minimum detection amount of Stx2e was 10~2copies/μL,which was 10times to 10000 times more sensitive than ordinary PCR.Several common porcine pathogenic bacteria such as Streptococcus,Salmonella,Haemophilus parasuis and Staphylococcus aureus were used as negative control for specificity test,and the results showed that only the positive standard has amplification signal in all five methods.The coefficient of variation of the intra-assay coefficients of variation were all below 3%,and the inter-assay coefficients of variation were below 4%,showing good repeatability.On the basis of the single-plex fluorescent quantitative PCR method,a triple fluorescent quantitative PCR method that can simultaneously detect three kinds of fimbriae genes of F4,F5,and F6 was established.The reaction conditions were optimized and the standard curve was established,and the correlation coefficient R~2was greater than 0.980,indicating a good linear relationship.The sensitivity test results showed that the minimum detection amounts of F4 and F6 fimbriae were 10~0copies/μL and 10~1copies/μL,respectively,which is 100 times more sensitive than ordinary PCR;the minimum detection amount of F5 fimbriae was 10~1copies/μL,which is more sensitive than ordinary PCR 1000 times,showing high sensitivity.The results of the specificity experiment showed that all the three fimbriae positive controls produced amplification signals,and there is no amplification curve for other bacteria,indicating strong specificity.The coefficients of variation of the intra-assay and inter-assay repeatability tests of the established triplex fluorescence quantitative PCR method were all below 5%,indicating good repeatability.A total of 88 samples collected from pig farms were detected by five established single-plex q PCR methods and triple q PCR methods,including 59 samples suspected of swine edema disease and 29 samples of piglet diarrhea.The results showed that there were 16 positive samples of Stx2e,7 positive samples of LT,2 positive samples of F4fimbriae,and no positive fimbriae of F5 and F6.The detection results have a high coincidence rate with the ordinary PCR detection results,indicating that the five virulence factors of pathogenic E.coli can be effectively detected,which can be used for the detection of clinical samples.In conclusion,this study successfully established five single-plex fluorescence quantitative PCR method for F4,F5,F6 fimbriae genes,LT genes,and Stx2e genes of pathogenic E.coli.At the same time,a triple fluorescent quantitative PCR detection method that can detect three kinds of fimbriae was also established.The established methods has strong specificity,high sensitivity and good stability,and provides technical means for the quantitative detection of virulence factors of piglet colibacillosis in clinical samples.