Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR for detection of porcine rotavirus
Specific probes and primers were designed according to the conserved region of porcine rotavirus VP6 gene sequence to establish a specific,sensitive,universal and efficient real-time fluo-rescence quantitative RT-PCR assay for PoRV detection.The amplified target fragment was 173 bp,and the reaction procedure was optimized with the constructed recombinant plasmid pMD19-T-VP6 as the template.The results show that the standard curve of this method is y=-3.113 9x+39.298,R2=0.993 7,and E=109.5％.The results were negative when the method was used to detect the pathogens of common infectious diseases in pigs.The minimum detection limit of standard quality particles was 1.32 copies/μL.The coefficient of variation within and between batches were less than 0.600％and 1.300％,respectively.The method had strong specificity,high sensitivity and good repeatability.114 samples for clinical diarrhea were detected,and the positive detection rate of this method(39/114)was better than that of conventional RT-PCR(22/114).All 39 positive samples were identified as PoRV by sequencing,and 22 positive plasmids were successfully constructed after VP7 gene was amplified.The sequencing results showed that 6 types of G-type PoRV were detected,and the detection rates were 9.09％(G3),22.72％(G4),18.18％(G5),31.82％(G9),4.54％(G11)and 9.09％(G26),respectively.The above results indicated that an universal TaqMan real-time fluorescence quantitative RT-PCR method has been established in this experiment,which is of great sig-nificance for the diagnosis,pathogen monitoring and epidemiological investigation of porcine rotavirus.
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