The Separation And The Development And Application Of Detection Method By Fluorescence RT-PCR Of Yak Rotavirus

Rotavirus?Rotavirus,RV?is one of the major pathogens causing yak diarrhea,which impacted the development of Yak breeding.the VP6 gene is the main target of molecular detection,but sequence of yak RV variation leads to the result that the RT-PCR method of Bovine Rotavirus?BRV?is not ideal for the detection of yak source RV,Seriously restricting the prevention and control of the disease.Therefore,the purpose of this study is to isolate the virus from positive samples,as well as to develop an insulated isothermal RT-PCR?iiRT-PCR?kit for on-site detecting yak RV and a quantitative RT-PCR method for epidemiological investigation of yak rotavirus in Northwest Sichuan Province.The results were as follows: 1.The separation and identification of yak rotavirusPositive samples of the yak RV were isolated and identified by MA-104 cells.A total of 10 strains were isolated and cultured in our study with MA-104 cell.Five of the samples were from Shangri-La,Yunnan,and five samples were from Yushu,Qinghai.They were had been cultured for tenth generations,and only one showed Cytopathic effecy after six generation.Each generation of cell virus was verified by RT-PCR and sequencing to be the RV and the isolated virus was identified as rotavirus by indirect immunofluorescence assay.This result laid a foundation in order to further pathogen and vaccine study of yak RV.2.The development and application of insulated isothermal RT-PCR?ii RT-PCR?Kit for on-site detecting yak rotavirusQinghai-Tibet Plateau covers a vast territory and it is thinly populated.In order to meet the needs of on-site detection of yak RV,a pair of primers and a fluorescent TaqMan probe were designed according to BRV and Yak RV VP6 gene sequence.After optimizing the reaction system and nucleic acid extraction by a commercial PetNAD nucleic acid extraction kit,The insulated isothermal PCR?iiRT-PCR?assay for on-site detecting bovine rotavirus?BRV?and yak RV was successfully established.The results showed that the assay could amplify specific fragment of BRV and yak RV,with no amplification of other unrelated pathogens,including Bovine Coronavirus,Bovine Astrovirus,Bovine Viral Diarrhea Virus,Escherichia Coli,Salmonella,Eimeria and Cryptosporidium.The detection limit of viral nucleic acid of the assay was 96.6 copies·?L^-1with good reproducibility.The results of the comparison with the reported detection methods show that the method is better than the reported method of detecting BRV and yak RV.Further,the yak RV of diarrhea cases in Northwest Sichuan plateau in 2016 was detected by the assay developed in this study,which proved that the assay is an choice for yak RV on-site detection.The established iiRT-PCR assay in this study was specific,sensitive and reproducible,and it can be used for detecting not only yak RV but also BRV.It takes only 1 hour from nucleic acid extraction to report test result with easy operation.On this basis,the reagent was assembled into the test kit with PetNAD after freezed and dried.Which makes it a valuable tool for rapid on-site diagnostics of Yak RV.3.The epidemiological investigation of yak rotavirus in Northwest Sichuan ProvinceAccording to the pair of primers designed by the assay,the real time SYBR Green quantitative RT-PCR method was established for detecting yak RV.The melting curve analysis showed one specific peak with a melting temperature?Tm?of 78.75 ? ± 0.15 ?,and no primer-dimers peak represented.No amplification was detected by this assay from unrealated DNA and RNA samples.The limit of detection of the reaction was 23.9 plasmid copies·?L^-1 of initial templates.the standard curve generated had a good linear relationship?r2?of 0.9981 and efficiency of 95.6% between initial templates numbers and Ct values.Good reproducibility was obraind for detecting con structed positive plasmid DNA with intra-assay variation of 0.01%⁰.08% and inter-assay variation of 0.02%⁰.06%.With a remarkable detection rate in yak RV,the the real time fluorescence quantitative RT-PCR method provided an useful tool for epidemiological investigation and quantitative analysis of the infection of yak RV.The epidemiological investigation in 88 samples of yak diarrhea from 14 pastures in Northwest Sichuan plateau in 2016 was 98.86%.The result shows that RV infection is an important cause of yak diarrhea in the area.