Establishment And Preliminary Application Of A Multiplex RR-PCR Assay For Identification Of Porcine Kobuvirus, Porcine Astrovirus And Porcine Kobuvirus

The raw pork which caused diarrhea in pigs by viral infection, and gradually attracted people’s attention which be transmitted to humans through market circulation, especially those not full studied virus which were new discoveried in recent years. Nowadays porcine kobuvirus (PKV), porcine torovirus (PToV), porcine astrovirus (PAstV) had a high detection rate of diarrhea in pigs, and other viruses which belong to them, it has been reported in a patient with diarrhea or intestinal found. These potential risks to human existence increased research value of PKV, PToV, PAstV. But the three viruses are difficult to distinguish about clinical and pathological, because their epidemiological and clinical symptoms are very similar. In order to further study of these three viruses which may caused diarrhea in pigs, we established a multiplex RT-PCR detection method which can detect these three viruses at the same time, sampling and preliminary testing the pork and pig intestines contents which from the slaughterhouse of Sichuan.The main results are as follows:1. The development of multiplex RT-PCR detection methods of PKV, PAstV and PToVAccording to the PKV 3D gene, PAstV ORF2 gene, PToV M gene, we designed three pairs of specific primers by Primer 5.0 software. Amplification of expected size were 358bp,938bp and 718bp fragments, respectively. Firstly, we established single RT-PCR detection technology for each virus, then established to detect a multiplex RT-PCR method PKV, PAstV and PToV by optimized reaction conditions and procedures simultaneously. The method can be amplified three kinds of specific bands at the same time; then compared with the multiplex RT-PCR and single RT-PCR methods. The multiplex RT-PCR detection method capable of detecting the lowest concentration PKV, PAstV and PToV are about 1.51×104copies/μL,1.19 ×103copies/μL,1.26×104copies/μL of sensitivity tests; the same template by five testing, the results were consistent with the single RT-PCR. It showed that this method had good specificity, sensitivity, and repeatability; both fast and accurate, so it can be used for PKV, PAstV and PToV detection.2. The multiplex RT-PCR detection method of preliminary applicationUsing the new method to detect 20 parts of pork from slaughterhouses, the results were all negative, and detected 96 parts of pig intestines contents from a slaughterhouse by the new method, the results of the statistical analysis found that PKV, PAstV, PToV positive detection rates were 47%,15%,11%,23 parts of mixed infection,12 parts detection PKV and PAstV, five copies of PKV and PToV mixed infections, four PAstV and PToV mixed infections, two samples detect three kinds of viral infections simultaneously. The established could be used to monitor or detect cases of clinical pigs, slaughter pigs and infected pork. To provide a reference method and theoretical basis for the protection of the healthy breeding pigs and safety of the pork.