Study Of The Standard Serological Methods In Detecting Virus For SV, Reo 3, MHV, MAd And RCV

Laboratory animals are indispensable for life science research in the experimental materials. Quality control of laboratory animals is an important guarantee for the success of animal experiments, in which virological monitoring is crucial content. In order to improve the accuracy and stability of viral test result, 5 kinds of viruses that should be tested in SPF mice or rats were selected to prepare standard anti-sera. At the same time, we have first respectively established ELISA kits for Sendai virus and reovirus type 3 for references of other viruses. For the pathogen detection, the RT-PCR techniques were successfully applied for Sendai Virus, Reovirus type 3, Mouse Hepatitis Virus, Mouse Adenovirus and Rat Coronavirus.To prepare standard anti-sera against viruses for quality control of viral detection on laboratory mice or rats, the viruses isolated from infected tissue were used to immune SPF BALB/c mice or Wistar rats, then sera were harvested and identified by IFA, IEA or ELISA. In this experiment, the stocks of anti-sera were prepared. A total of 18mL of mouse anti-SV(IFA 1:640), 18mL of mouse anti-Reo-3(IFA 1:640), 25mL of mouse anti-MHV(IFA 1:320), 18mL of mouse anti-MAd(IFA 1:320) and 15ml of rat anti-RCV(IFA 1:160) were prepared. And there were no cross-reaction among the anti-sera separately.In this paper, ELISA systems for anti-SV and anti-Reo-3 were established. Plenty of virus antigen were amplified in cell culture, controlled with the normal lysates of cell culture. The best working density was titrated and the results showed high accuracy, sensitivity, stability and specificity for these viruses detection comparing with the results by IFA method.The best working density of SV antigen and the SV-anti-serum were 2.5μg/mL and 1:6,000 respectively, the inter-assay coefficient of variation of special antigen is 7.9%; the intra-assay average coefficient of variation is 4.9%. The detection sensitivity is 1:25,600. There is no cross-reactivity with Reo3, MHV, MAd, Vaccinia Virus and POLY. The stability test showed the relative deviation is below 20% within 2 days in 37℃.The best working density of Reo-3 antigen and the Reo-3-anti-serum were 5μg/mL and 1:2400 respectively, the inter-assay coefficient of variation of special antigen is 3.8% respectively; the intra-assay average coefficient of variation is 4.0%; the detection sensitivity is 1:4400. There is no cross-reactivity with SV, MHV, MAd, Vaccinia Virus and POLY. The stability test shows the relative deviation is below 27% within 2 days in 37℃.To establish the RT-PCR method for detection of pathogen, we extracted RNA from supernatant of viral culture or infected tissue, optimized the amplification conditions of RT-PCR and got the target products of SV, MHV,Reo-3 and RCV respectively. But no products had been amplificated by RT-PCR from positive serum samples from mice.In this experiment, the immunized anti-sera were prepared with high titer, and were sensitive and specific to each virus detection, it could be used widely as the standard control sera to detect virus serologically or detect pathogen. And the ELISA methods for SV and Reo3 were ideal in repetition, stability, specificity, and sensitivity.Stock anti-sera were prepared as positive control in this study, improved the shortage of by using positive serum from infected mice as control. It lays the foundation for establishing standard reagent. At the same time, we establish ELISA and RT-PCR as additional comparative techniques to IFA and IEA, it should be a good way for establishment of detection platform in laboratories.