| Rotavirus(RV) who belongs to Reoviridae family is the main etiology cause of infant and a variety of young animal diarrhea. The pathogenicity of RV is extremely high with nearly to 100% mortality rate in the piglets younger than 7-day old. The incubation period of RV is short and the infectivity is strong. Once part pigs were infected, the disease would spread rapidly, causing a massive outbreak, bringing huge economic losses to swine industry. Recently, conventional RT-PCR method was the only detection method for clinical detection of rotavirus. While this method has low sensitivity and is hard to use for early detection. The quantitative RT-PCR method with 100-1000 times higher sensitivity can solve this problem.In order to establish a sensitive and specific G9 genotype porcine rotavirus(PoRV) TaqMan fluorescence quantitative RT-PCR detection method, and provide effective technical means for the clinical diagnosis of rotavirus, a pair of probe and primers was designed based on the conserved region of VP7 gene of PoRV(G9) strain NMTL. Under the optimized conditions, the standard curve was set up by a serious 10-fold dilutions of the positive plasmid, which was built by the connections of VP7 gene fragments and pMD18-T carrier, and the method was evaluated by testing the specificity, sensitivity and repeatability. The standard curve showed a certain linear relationship between Ct value and the amount of template DNA, linear equation for Y=-3.436X+41.70, and the linear correlation coefficient was R2=0.991. This assay was specific for G9-genotype PoRV detection, and had no cross-reactions with other related porcine pathogens such as PoRV(G5-genotype), transmissible gastroenteritis virus(TGEV), porcine epidemic diarrhea virus(PEDV), porcine reproductive and respiratory syndrome virus(PRRSV) and porcine cirovirus type 2(PCV2); Moreover, the detection limit of the assay was 33.6copies/μL, which was 1000 times more sensitive than that of conventional RT-PCR. The results of research suggested that the method showed good specificity, high sensitivity and good repeatability, which could make judgments and quantify to the rotavirus in the early infected piglets, and played an important role in preventing mass outbreak of rotavirus.Pathogenicity of PoRV NMTL strain was tested on 3-day healthy newborn piglets. 8 piglets were selected and random divided into two groups, the control group and the test group. Each piglet in the test group was challenged with 5×105 TCID50 NMTL strain by oral infection. Then, the two groups were fed separated in same condition. One piglet in challenge group showed diarrhea early 8 hours post challenging. The number of diarrhea piglets was increasing as time goes on, manifesting vomiting, dehydration, weight loss and other symptoms such as watery and yellowish stool as well as dispirited spirit. The earliest death was happened in 36 h post challenging, and dead piglets appeared purple skin with obvious symptom of dehydration. The anal swab samples were detected by the developed TaqMan quantitative RT-PCR of G9 genotype PoRV every 12 hours post infection. The results showed that the virus copy number increased with the increase of attack time, and the copy number reached the highest value in 60-84 h. Pathological histology analysis revealed that the small intestine of the piglets in the challenging group had severe pathological change, such as, atrophying and coarsening of duodenum, jejunum and ileum; necrosis and shedding of chorionic villus; vacuolization of the mucosal epithelial cells of the villus; edema of lamina, While there was no obvious pathological changes in the control group.This study established an effective technical method for the clinical diagnosis of rotavirus, which provided a material and theoretical basis on the research and development of rotavirus vaccine and pathogenesis. |
