Establishment Of RT-PCR Detection Assay For Porcine Kobuvirus And Application

Porcine Kobuvirus was first detected in2008. It has been verified as a genotype of Kobuvirus according to the homology of Aichi virus, an RNA virus which may cause the diarrhea of piglets. This virus are widely infected in each countries of the world and the impact to pig industry should not be ignored. In this study, in order to prevent and control the disease we committed to find a method for detecting Kobuvirus in porcine with efficient, accurate and stable. Acorrding to the nucleic acid sequences of porcine Kobuvirus S-1-HUN strain published on Genbak, designed a pair of specific PCR primers to amplify a target fragments which has a length of313bp and located at3D-RNA polymerase region. Collecting samples from different scale pig farms in Sichuan Province. Extract the viral RNA from the stool of diarrhea piglets, by reverse transcription to cDNA and optimization of the reaction condition to establish a new RT-PCR method for detecting Kobuvirus. Recovery DNA product after reaction, then recombinant plasmid and sequencing positive bacteria, the similarity between reaction product and target fragment is necessary to ensure the accuracy of this method. Nine different pathogens including PRRSV, PRV, Pasteurella are used for experiment of specific, which could be the Identified causes of diarrhea of piglets. Detect the same sample by the established assay with these pathogens to ensure the specificity of this assay.10-fold serial dilution to the RNA solution and then experiment with each concentration in order to find a minimum content of RNA virus that can be detected in samples. Detected Kobuvirus in123stool samlpes from difference regions in Sichuan Povince by this RT-PCR assay. The assay results prove two conclusions: The positive rate of collected samples is up to59.3%and basically reflects the infection of large-scale pig farms in the Province. We can also find that the RT-PCR assay method which established by this study has good repeatability, sensitivity and specificity for large-scale clinical testing. The efficiency is better than the assay of universal primers for detecting porcine Kobuvirus.