Establishment And Preliminary Application Of TaqMan Multiplex QPCR Method For Detecting Common Four Viruses And Four Bacteria In Swines

The diagnosis of swine fever virus,porcine reproductive and respiratory syndrome virus,porcine circovirus type 2 and pseudorabies virus in pigs is difficult to diagnose because of similar clinical symptoms,Staphylococcus aureus,Streptococcus,Salmonella,and Escherichia coli are threatening animal health and public health safety all the time,and they often have secondary infections in viral infections,furthermore,the cases of two to three mixed infections of four viruses and four bacteria are more common.Therefore,to establish a specific,sensitive,rapid and quantitative detection methods for clinical diagnosis and detection of diseases is necessary,while can provide technical support for the prevention and purification of disease.In this study,TaqMan multiplex qPCR method was established to detect aforementioned four viruses and four bacteria by designing specific primers and TaqMan probes for the conserved sequences registered in NCBI,and was preliminary applied in clinical practice.The study provided a good qPCR method for the diagnosis and laboratory research of common diseases in pigs,layed a certain technical basis for disease prevention and control.The study obtained the following results:1.Four pairs of specific primers and four probes were designed for the E2 gene of swine fever virus,the ORF7 gene of porcine reproductive and respiratory syndrome virus,the ORF1gene of porcine circovirus type 2 and the gE gene of swine pseudorabies virus,a Taq Man multiplex qPCR method capable of detecting four viruses simultaneously was established and its specificity,sensitivity and reproducibility were tested.The results showed that the standard curve of the method had a good linear relationship;the specificity was strong that there was no cross-reaction between different viruses and only the positive template had an amplification signal;the detection limit respectively was CSFV 3.28×10~2 copies/μL,PRRSV 3.76×10~2copies/μL,PCV2 4.74×10~2 copies/μL,PRV 2.87×10~1 copies/μL,the sensitivity was at least 10times higher than that of conventional PCR;the repeatability was good because the coefficient of variation was less than 5%.Sixty-seven clinical samples were tested using the method,one more positive and one mixed samples were detected than conventional PCR.2.To establish TaqMan multiple qPCR methods that the ability to detect four bacteria simultaneously,four pairs of specific primers and four probes were designed for the nuc gene of Staphylococcus aureus,the ef-tu gene of Streptococcus,the ivnA gene of Salmonella,and the 23S rRNA gene of Escherichia coli,and their specificity,sensitivity and reproducibility were tested.The results showed that the established method had a good linear relationship,and the specificity is strong;the detection limit respectively was Streptococcus 1.08×10~2 copies/μL,Staphylococcus aureus 9.59×10~1 copies/μL,Salmonella 8.55×10~1 copies/μL,E.coli 5.82×10~2copies/μL,the sensitivity was 10 times higher than conventional PCR;the repeatability was well that the coefficient of variation of repeated tests were below 3%,four kinds of pathogens can be specifically detected using the method to detect artificial infections.