| Porcine rotavirus (PoRV) is a kind of highly contagious enteric infectious diseases which can mainly cause vomiting, diarrhea, dehydration and weight loss. Even it also can cause the piglet death, especially for about a week old of piglets. The disease, which mainly breaks out in the winter and early spring when it is cold, spreads rapidly and can bring huge losses to the pig industry. Currently, the main method to detect PoRV is conventional RT-PCR which is less sensitive compared to the fluorescence quantitative RT-PCR. Conventional RT-PCR can only detect the onset of severe period of pigs, while for latent infection or the early stage of infection of pigs need more sensitive detection method for etiological diagnosis, in order to diagnose PoRV promptly and take effective control measures to reduce the losses caused by the outbreak of PoRV.This study is mainly to establish a more sensitive TaqMan fluorescence quantitative RT-PCR method for the detection of PoRV. A pair of conventional RT-PCR primers and a pair of fluorescence quantitative RT-PCR specific primers and probe were designed based on the VP6gene sequences of the reference porcine rotavirus (PoRV) strain available in the GenBank database. A recombinant plasmid pMD18T-VP6containing the target gene was constructed as a standard control, and then a TaqMan fluorescence quantitative RT-PCR assay for the detection of PoRV was established. The specificity, sensitivity and reproducibility of the assay were evaluated and compared with conventional RT-PCR. From the standard curve, we found the concentration of the reaction templates had a good linearity relation with the fluorescent quantitative CT value. The equation of the straight line was y=-3.979gx+43.152and the correlation coefficient reached0.997. The detection limit achieved4.6×102copies/μL. By dilution analysis, the real-time RT-PCR was100times more sensitive than the conventional RT-PCR. No cross-reactions were found with the samples containing classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus of swine (TGEV), and porcine epidemic diarrhea virus (PEDV), swine pseudorabies virus (PRV), porcine circovirus (PCV2). The results showed that with high sensitivity, specificity and reproducibility the new method can take a series of steps to control validly so as to reduce the losses of PoRV outbreak in the early infection.Using porcine rotavirus TM-a strain isolated by our laboratory to infect3-day-old colostrum-deprived piglets. The strain’s TCID5Owas10-658/0.1ml and the load of PoRV was1.8μ106copies/μL in MA-104cell cultures detected by this new method. In all,16 PoRV negative pigs were used, which were split into four groups:blank control group, low dose group infected with4μ106TCID50, the middle dose group infected with2×107TCID50and high dose group infected with1×10TCID50. This study successfully established a rotavirus infection models in piglets cases. The clinical symptoms indicated that diarrhea symptoms appeared earlier with the increase of the infection dosage. The piglets developed watery diarrhea after10h infection. By the method of fluorescence quantitative RT-PCR we detected the pathogen in faeces18hours after infection and the load of PoRV in faeces was increased increased with time. By HE experiment, we could found that there were severe pathological changes on the small intestines of the infected piglets, with intestinal villi atrophy or fall off, while the blank control group were unchanged. Through immunohistochemical method we can see that the virus generally existed in the small intestine. Based the copy number we detected in the faeces by the method of fluorescence quantitative RT-PCR, we researched the law of expelling of virus of pigs infected PoRV. The above work provided a theoretical and material basis for the future research and development of diagnostic reagents, the new vaccine and vaccine effectiveness evaluation. |
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