Preparation The Standard Serum Against TMEV,Ect,LCMV,POLY,PVM And The Study Of Detection Methods With ELISA

The virus detection of laboratory animal is an important tool to ensure high qulity of laboratory animal and accurate results of animal experiments. At present, IFA and IEA are frequently used for virological detection in our laboratory. We establish an ELISA method as an additional technique to strengthen the result comparison. This study focused on the preparation of the standardized serum and the establishment of the ELISA and PCR detection methods, and our goal is to improve the detection system of our laboratory and shorten the gap between domestic and foreign countries.Theiler's mouse encephalomyelitis virus(TMEV),Ectromelia virus(Ect),Lymphatic choriomeningitis virus(LCMV),Polyoma virus(POLY) and Pneumonia virus of mice(PVM) were chosen in this study and these pathogens must be excluded in clean and SPF graded laboratory animals.The positive control sera of TMEV,Ect,LCMV,POLY and PVM were obtained by immunizing BALB/c mice. And the sensitivity and specificity of the harvested sera were evaluated with IFA and IEA when their titre reached a certain requirement. As a result, five kinds of serum titers were 1:640,1:320, 1:640,1:320, 1:1280,1:640, 1:640,1:640, 1:640. 1:320 with IFA. IEA, respectively. We obtained high quality of immunized sera with high sensitivity and specificity and are available as the standard control sera in the virus detection of laboratory animal.This study has established the ELISA method for detection of TMEV and Ect antibody. BHK-21 cell line was infected by TMEV and Vaccinia virus to prepare specific virus antigen. The optimal working concentration of the coated antigen and the titration of standard positive were determined, the coated antigen and the positive serum were 2.5μg/mL and 1:200, respectively. Experiments were carried out to evaluate sensitivity, specificity, repeatability and stability. The detection sensitivity by our ELISA is 1:3200 dilution; There is no cross-reactivity with Pneumonia virus of mice, Mouse hepatitis virus, Sendai virus and Reovirus-3. Inter-assay and intra-assay coefficients of variation of spectial antigen and normal antigen are less than 10%, and the stability test showed the relative deviation is less than 20% at 37℃after 2 days. In our study, the method of detection TMEV and Vaccinia virus with ELISA showed more sensitive, specific, repeatabe and trustable. The result of comparison between ELISA kits indicated the related sensitivity is 100% for TMEV and 92.9% for Ect, coincidence is 90.48%,93.3%. Therefore, ELISA we created could be used to detecte the TMEV and Ect antibody in laboratory animals.In the mean time, we tried to establish the nested RT-PCR or PCR methods for detection LCMV and POLY pathogen, respectively. A 149bp specific fragment was amplified with nested RT-PCR for LCMV. The PCR products were cloned and sequenced for the homologous comparison with nucleotide sequence of LCMV in Genebank by BLAST on line.The data indicated 99.32% similarity; The method of PCR was performed by using a pair of specific primers that amplified a 272 bp specific fragment to detection POLYIn conclusion, the new ELISA and PCR techniques enriched the monitoring methods for virological detection, and will be useful in virological detection in laboratory animals.