Establishment And Application Of Multiple RT-qPCR Detection Methods For The Main Pathogens Of Piglet Diarrhea In Jilin

Porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis of swine virus(TGEV)and porcine rotavirus(Porcine rotavirus,Po RV)are the main viruses that cause diarrhea in piglets.Using PEDV's M gene,TGEV's N gene,and Po RV's VP7 gene as target genes,design specific primers and probes labeled with different fluorophores to establish multiple real-time fluorescent quantitative PCR detection methods for these three viruses,and carry out specificity,sensitivity and repeatability test.The results show that this detection method is highly sensitive,and the detection limits for p MD18-PEDV-M,p MD18-TGEV-N and p MD18-Po RV-VP7are 3.91×10~2copies/?L,8.39×10~1copies/?L and 4.54×10~2copies/?L,respectively.The sensitivity is 10 times higher than that of conventional PCR;only PEDV,TGEV,Po RV c DNA and the positive control have specific amplification curves with good specificity;the coefficient of variation within and between groups is less than 5%,with good repeatability.Using this detection method to detect clinical samples,the positive rates of PEDV,TGEV and Po RV were 52%,0.9%,and 1.5%,respectively,which were 10more positives and 4 positives for mixed infections than conventional PCR.E.coli,Salmonella and C.perfringens can cause bacterial piglet diarrhea.In order to establish a method for simultaneous detection of swine E.coli,Salmonella and C.perfringens,specific primers and Taq Man probes were designed for E.coli irp2 gene,Salmonella fim Y gene,and C.perfringens plc gene respectively.The reaction conditions were optimized,and a multiple real-time fluorescent quantitative PCR detection method for simultaneous detection of 3 kinds of bacteria was established.The results showed that the minimum detection limits of this method for the plasmid standards of E.coli,Salmonella and C.perfringens were 9.28×10~3 copies/?L,3.09×10~3copies/?L and 4.37×10~3copies/?L,respectively.The lowest sensitivity is 10 times higher than conventional PCR;it only specifically amplifies E.coli,Salmonella and C.perfringens.It has no amplification curve for some other bacteria and has strong specificity;the coefficient of variation within and between groups is less than 5%,with good repeatability.Using this detection method to detect clinical samples,the positive rates of E.coli,Salmonella and C.perfringens were 55.4%,5.4%,and 7.1%respectively,which were 6 more positives than conventional PCR.Based on the genetic evolution analysis of PEDV M,TGEV N,Po RV VP7 genes,the nucleotide and amino acid homology of the genes were analyzed,and the NJ phylogenetic tree was constructed.The results showed that 11 PEDV M genes were successfully amplified,all of which were G2 type strain;1 TGEV N gene;2 Po RV VP7genes,all of which are G9 genotype.The results showed that PEDV M,TGEV N,and Po RV VP7 gene sequences are highly conservative and can be used as target genes for virus infection detection.The epidemic strain of PEDV in Jilin area is of type G2,which is far from the G1 vaccine strain CV777.The epidemic strain of TGEV is closely related to the vaccine strain Hua virus strain H.The epidemic strain of Po RV is of type G9,which is separate from the G5 vaccine strain.It belongs to different serotypes,which may lead to unsatisfactory vaccine immunity.