Establishment And Preliminary Application Of Detection Methods For Murine Hepatitis Virusand Tyzzer’s Organism

The quality and level of the entire experimental studies will beaffected by laboratory animals which is one conditionality factor of the fourbasic elements in life science research. Quality of laboratory animals is soimportant to make sure the accuracy and repeatability of animalexperiments in scientific research. Since1994, a number of experimentalanimal’s standards were issued to ensure our animal management industryto legalization and standardization. The detections of Mouse hepatitis virusand Tyzzer’s organisn were common test items in laboratory animals’microbiological examinations.Currently, enzyme-linked immunosorbent assay andimmunofluorescence experiments were the master detecting method ofMouse hepatitis virus and Tyzzer. They with a low rate of detection ratewere invalid in early infection and immunodeficiency.PCR amplification and reverse dot blot hybridization technology orSNaPShot were combined in this study. According to the genomicsequence of mouse hepatitis virus and Tyzzer, specific primers and probeswere designed. A series of experimental conditions were optimized. It wasestablished and applied that the reverse dot blots hybridization detectionmethods of Mouse hepatitis virus and Tyzzer. And a new rapid, sensitiveand specific SNaPshot method was established to genotype MHV1, MHV3,JHM and A59of mouse hepatitis virus. The contents in this study consist of part1and part2.Part1Establishment and preliminary application of reverse dotblot for the detection method of Murine Hepatitis Virus andTyzzer’s organism.Section1Establishment and preliminary application of reverse dotblot for the detection of Murine Hepatitis VirusObjective To establish a method for detection of MHV by reverse dotblots (RDB). Methods L929cell was infected with four MHV strainsrespectively. MHV was harvested and extracted of RNA for reversetranscription to cDNA. Primers and specific probes were designed on thebasis of conserved sequence of MHV and forward primer was labeled withbiotin. The method of reverse dot blot was developed followed bypolymerase chain reaction (PCR). In the same time, its stability, specificityand sensitivity were detected. In addition,41mice were detected by RDBand ELISA. The positive samples tested by RDB were detected by cloneand sequencing. Results It could be keep the test in4℃for8months. Thespecificity of RDB was100%and for MHV, Salmonella sp.,Staphylococcus aureus and Hepatitis B virus pointed to a positive test forMHV with limit of detection was down to5ng/μL. In41mice tested byRDB, there were five MHV positive mice distribution in three groups. Butby ELISA, there were five MHV positive mice distribution in two groups.The results by cloning sequencing and RDB for positive samples wereconsistent. Conclusions The reverse dot blots can simplicity, reliable,sensitively and specifically identifies MHV of laboratory animals. Section2Establishment and preliminary application of reverse dotblot for the examination of Tyzzer’s organismObjective To establish a simple, stable, specific and sensitive methodfor detection of Tyzzer’s organism by reverse dot blotting (RDB). MethodsPrimers and specific probes were designed according to the conservativesequence of Tyzzer16S rDNA. Forward primer was labeled with biotin.The reverse dot blotting method was established followed by PCRamplification. The specificity and sensitivity of this method weredetermined. Next,41mice and38rats were examined by RDB, ELISA andIFA. Results The RDB method showed a high specificity, and in the testingof the79laboratory animals, its limit of detection was4.5ng/μL.Compared the results of ELISA and IFA, its consistence with ELISA was100%and the positive rate was7.95%(6/79), the consistence with IFA was92.4%(73/79), and the positive rate was0%. Conclusions An accurate,sensitive and specific method in combination with PCR and RDB indetection of Tyzzer’s organism is established in this study.Part2Examination of application SNaPshot typing assays formouse hepatitis virus.Objective To establish a new detection for typing assays of fourstrains included MHV1,MHV3,JHM and A59.Methods Based on the resultsof four strains sequence alignment, two pairs of PCR amplified primers andfour single-base primers were designed. RNA of four MHV strains wasextracted for reverse transcription. The SNaPShot assay was developedafter polymerase chain reaction (PCR) and purification. MHV genotypeswere analyzed by capillary gel electrophoresis. SNaPshot assay was optimized; its sensitivity and specificity were analyzed. Furthermore,41mice serum samples were detected by ELISA and SNaPshot. The positivesamples tested by ELISA and SNaPshot were detected by cloning andsequencing. Results The best conditions of SNaPshot test were T1~T4primers modified by0,3,10and15poly T, the concentration proportionbeing1(2μmol/L):1(3μmol/L):1(2.5μmol/L):1(5μmol/L) and the size ofprimers being16bp,19bp,26bp and31bp. The detection limit of SNaPShotassay was1.25ng/μL (MHV cDNA). The specificity of SNaPShot was100%. Its accuracy was100%(41/41) compared with ELISA, cloning andsequencing; the positive samples were JHM. Conclusions An sensitive,specific and accurate SNaPshot detection for typing of MHV1、MHV3、JHM、A59had been established.