Optimization Of Nucleic Acid Detection Methods And Comparative Analysis With Results By Serological Methods Of Three Viruses In Mice

Laboratory animal is an important instrument for life science research.Their own quality is very important,especially the quality of microorganism.So it is necessary to monitor the microorganism quality of laboratory animal.At present,viruses screening of laboratory animals mostly employs serological methods for retrospective investigation of past infections of animals.However,pathogeny detection should be used for the pathogenic present status of animals in the current period and in the surrounding environment.The pathogeny detection technique based on nucleic acid amplification has high sensitivity and specificity.And it can be used in pathogeny detection of laboratory animal.However,in the pretreatment process of samples,the influence of storage and transportation conditions of samples on nucleic acid detection is unreported.For the moment,nucleic acid detection method for virus detection of laboratory mice is seldom used in our country,and its applicability to the health monitoring of laboratory mice remains to be explored.Therefore,this paper studied the effect of storage condition of mouse samples on the detection of three virus nucleic acid and applied the nucleic acid detection method to detect samples collected by our testing laboratory to carry out the research on the suitability of nucleic acid detection method.First,this paper studied the effect of sample storage temperature and different types of preservatives on the detection of three viral nucleic acids.At room temperature(25?)and low-temperature(4?),the samples were kept with saline and Buffer AVL as preservatives respectively.The amount of MHV,Reo-3,and MNV nucleic acids detected by fluorescence quantitative PCR.The results showed that the sample in the Buffer AVL group nucleic acid degradation was lower.And in the storage condition 4? was better than room temperature(25?),the decrease in the amount of nucleic acid detected was less than 50%on the third day of samples stored at 4? with Buffer AVL.Therefore,low-temperature transportation and using Buffer AVL as the preservative can effectively protect the viral nucleic acid.Then,the results of nucleic acid detection and serological detection on mouse samples were compared.In 272 fecal contents samples and corresponding serum samples of mice,the nucleic acid detection rate of MHV,Reo-3,MNV was 17.3%,18.8%and 16.9%,while the serological antibody positive rate was 1.2%,1.2%and 14.8%,respectively.The results of nucleic acid detection were not completely consistent with the results of serological examination.Nucleic acid detection is complementary to serological method as a pathogen detection method in laboratory mouse pathogeny monitoring.This paper also explores the latest technology called NASBA-ELISA.The RNA of SV,MHV and Reo-3 served as template for NASBA amplification.Their amplified products were analysed by gel electrophoresis,and the bands were single and clear.But it failed to achieve products signal multiplication by ELISA.This method needs further optimization in laboratory applications.