实验动物是生命科学研究的重要工具，小鼠微小病毒（MVM）、小鼠肝炎病毒（MHV）、呼肠孤病毒 III 型（Reo-3）、小鼠诺如病毒（MNV）这4 种病原体是实验用小鼠最常见的易感染病原体，对实验动物自身的健康和实验结果的准确性有极大的威胁。基于以上原因，本研究结合重组酶介导链替换核酸扩增技术 （Recombinase-aid nucleicacid Amplification ，RAA）针对前文所述四种病原体建立了快速、简单易用的病毒检测方法。具体结果如下所示：

1建立了小鼠微小病毒（MVM）的荧光RAA检测方法

根据小鼠微小病毒（MVM）的NS1基因，设计并筛选特异性引物，建立了该病毒的荧光RAA检测方法。可区分MVM和其他5 种常见的实验小鼠易感染病毒及致病菌；检测方法灵敏度达10 copies/μL；45 份样本分别用荧光定量PCR检测方法和荧光RAA检测方法进行检验结果符合率为100%。

2建立了小鼠肝炎病毒A59型（MHV-A59） 的荧光RAA检测方法

根据小鼠肝炎病毒A59型（MHV-A59）的S蛋白基因，设计并筛选特异性引物，建立了该病毒的荧光RAA检测方法。可区分MHV-A59和其他5 种常见的实验小鼠易感染病毒及致病菌；检测方法灵敏度达10 copies/μL；60 份样本分别用荧光定量PCR检测方法和荧光RAA检测方法进行检验结果符合率为98.3%。

3建立了小鼠呼肠孤病毒 III 型（Reo-3）的荧光RAA检测方法

根据小鼠呼肠孤病毒 III 型（Reo-3）的L2突刺蛋白基因，设计并筛选特异性引物，建立了该病毒的荧光RAA检测方法。可区分Reo-3和其他9 种常见的实验小鼠易感染病毒及致病菌；检测方法灵敏度达10 copies/μL；45 份样本分别用荧光定量PCR检测方法和荧光RAA检测方法进行检验结果符合率为97.8%。

4建立了小鼠诺如病毒（MNV）的荧光RAA检测方法

根据小鼠诺如病毒（MNV）的 ORF2基因保守序列，设计并筛选特异性引物，建立了该病毒的荧光RAA检测方法。可区分小鼠诺如病毒（MNV）和其他9 种常见的实验小鼠易感染病毒及致病菌；检测方法灵敏度达10 copies/μL；53 份样本分别用荧光定量PCR检测方法和荧光RAA检测方法进行检验结果符合率为98.1%。

本研究基于重组酶介导链替换核酸扩增技术 （Recombinase-aid nucleicacid Amplification ，RAA），建立了四种实验小鼠易感染病毒的恒温扩增检测方法。该方法可以在39℃-42℃条件下完成对目的核酸片段的检测，具有速度快、特异性强、灵敏度高、操作相对简单等特点，在实验动物检测中具有广阔的临床应用前景 ，对实验动物微生物的诊断、治疗和疫情的控制具有重要意义。

Experimental animals are important tools for life science research. The four pathogens of mouse parvovirus (MVM), mouse hepatitis virus (MHV), reovirus type III (Reo-3) and mouse norovirus (MNV) are the most common infectious pathogens of experimental mice, which are dangerous to the health of experimental animals and the accuracy of experimental results. Based on the above reasons, this study has established a rapid, simple and easy-to-use virus detection method for the four pathogens mentioned above with Recombinase-aid Nucleic Acid Amplification (RAA) methord. The specific results are as follows:

1. RAA-Exo detection method for mouse parvovirus (MVM)

According to the NS1 gene of mouse parvovirus (MVM), a specific primer was designed and screened, and a fluorescence RAA detection method for the virus was established. It can distinguish between mouse parvovirus (MVM) and 5 common laboratory mice susceptible to infection with viruses and pathogenic bacteria; The sensitivity of the detection method reaches 10 copies/μL; 45 samples were tested by fluorescence quantitative PCR and fluorescence RAA respectively, and the coincidence rate was 100%.

2. RAA-Exo detection method for murine hepatitis virus strain A59 (MHV-A59)

According to the S gene of murine hepatitis virus strain A59 (MHV-A59), a specific primer was designed and screened, and a fluorescence RAA detection method for the virus was established. It can distinguish between MHV-A59 and 5 common laboratory mice susceptible to infection with viruses and pathogenic bacteria; The sensitivity of the detection method reaches 10 copies/μL; 60 samples were tested by fluorescence quantitative PCR and fluorescence RAA respectively, and the coincidence rate was 98.3%.

3. RAA-Exo detection method for mouse reovirus type III (Reo-3)

According to the L2 gene of mouse reovirus type III (Reo-3), specific primers were designed and screened, and a fluorescence RAA detection method for the virus was established. It can distinguish reovirus type III (Reo-3) and 9 common laboratory mice susceptible to infection with viruses and pathogenic bacteria; The sensitivity of the detection method reaches 10 copies/μL; 45 samples were tested by fluorescence quantitative PCR and fluorescence RAA respectively, and the coincidence rate was 97.8%.

4. RAA-Exo detection for murine norovirus (MNV)

 According to the ORF2 gene of mouse norovirus (MNV), a specific primer was designed and screened, and a fluorescence RAA detection method for the virus was established. It can distinguish between mouse norovirus (MNV) and 9 common laboratory mice susceptible to infection with viruses and pathogenic bacteria; The sensitivity of the detection method reaches 10 copies/μL; The coincidence rate of 53 samples tested by fluorescence quantitative PCR and fluorescence RAA was 98.1%.

Based on Recombinase-aid nucleicacid Amplification (RAA), this study establishes four kinds of recombinase-Aid nucleicacid amplification detection methods for susceptible virus infection of experimental mice. The method can complete the detection of nucleic acid fragments at 39℃-42℃, and has the characteristics of fast speed, strong specificity, high sensitivity and relatively simple operation. It has broad clinical application prospects in the detection of laboratory animals, and which also have big significance for the accurate diagnosis,  timely treatment and control of the epidemic situation of laboratory animals.