Isolation And Establishment Of A RT-PCR Detection Assay For Tupaia Rotavirus

Objective:To know experimental Tupaia rotavirus infection, the establishment of RT-PCR method for detection of Tupaia rotavirus.This method is fast and simple, Tupaia rotavirus molecular epidemiological studies for the future and develop the national quality standard of experimental, regulation and supervision of quality provides the basis. Provide valuable data for the study of rotavirus infection and related vaccines and drug development.Methods:1.This research collected 360 fecal samples and serum samples from wild Tupaia.2.The preliminary screening of rotavirus carried by the sample was carried by ELISA and colloidal gold.3.Use Triol method and humoral viral DNA / RNA Kit small amount(Axy Prep)extraction of viral RNA in the stool sample.4.According to 9 rotavirus genes, glycoprotein VP7 gene sequence of conservative area(Gen Bank M21843) design synthesis RT-PCR primers, the desired fragment(365bp) was obtained by agarose gel electrophoresis.5.The fragment was purified utilizing purification kit made of plastic recycling and sent to sequencing the gene sequencing company.6.The positive fecal samples were amplified and cultured by centrifugation and filtration and inoculated with MA104 cells.7.The specificity and sensitivity of RT-PCR detection for the detection of RNA in the viral liquid of the virus were detected.Results:1.By colloidal gold and ELSA method the 360 collected fecal samples and serum samples were not detected rotavirus positive.2.By RT-PCR method, a positive sample was detected, the fragment size was365 bp,the fragment was purified utilizing purification kit made of plastic recycling and sent to sequencing the gene sequencing company.Gene fragments sequenced by the NCBI BLAST alignment of sequences and the resulting strain of bovine rotavirus VP7 fragment nucleic acid sequence coverage of 92%, matching the consistency of99%.3.Positive sample were inoculated into MA104 cells were separated, successfully harvested from the first generation to the fourth generation of cellular virus solution.4.Application of this RT-PCR method respectively sequencing rotavirus, reovirus RNA samples, adenovirus, herpes simplex virus DNA sample as a template to amplify only the sample being measured appears target band. This 104-fold dilution of rotavirus RNA template, can still be detected in the target band. Proved that this method is very high specificity and sensitivity.Conclusion:1.By comparing the optimal initially established a rapid method for detection of Tupaia rotavirus infection, the detection methods and other pathogens such as call intestinal arc virus, adenovirus, simple herpes virus detection no cross reaction and good specificity. And the sensitivity is also higher.2.Successfully isolated a strain of Tupaia rotavirus by sequence alignment and bovine rotavirus strains of nucleic acid sequence similarity is highest for the future development of molecular epidemiology of rotavirus in Tupaia provides some reference.3.Provides fast, reliable method for detection of rotavirus infection of Tupaia. For the national quality standard experimental Tupaia, to provide corresponding basis for the regulation and supervision of the quality of Tupaia.