小鼠多瘤病毒荧光PCR检测方法的建立及其在裸鼹鼠感染情况调查中的应用

Establishment of a fluorescence quantitative PCR assay for murine polyomavirus detection and its use in the detection in naked mole rats

目的建立多瘤病毒荧光定量PCR检测方法,并对裸鼹鼠中多瘤病毒感染情况进行调查。方法比较NCBI中小鼠多瘤病毒(Genbank:NC_001515)的核酸序列,选择保守区域设计引物和探针,建立多瘤病毒的荧光定量PCR方法,对方法的敏感性和特异性进行验证。人工感染9只1日龄KM乳鼠,21 d后采集心、肝、脾、肺、肾、脑、胸腺、盲肠内容物和血液等组织脏器,用建立的荧光PCR方法进行检测,验证方法在应用中的有效性,最后用该方法检测62份裸鼹鼠的盲肠内容物样本。结果建立的检测方法特异性好,在以多瘤病毒为模板时出现明显的荧光信号,以猴空泡病毒、小鼠K病毒、小鼠微小病毒和大鼠细小病毒H-1株为模板时无荧光信号出现;方法的最低检出限为100 copies/μL;人工感染小鼠的心、肝、脾、肺、肾和盲肠内容物中均检测到多瘤病毒核酸,其中肺组织中含量最高,脑、胸腺和血液中未检出;62份裸鼹鼠的盲肠内容物样本经检测多瘤病毒为阴性。结论建立的多瘤病毒荧光定量PCR方法可有效检测动物组织中的多瘤病毒,对裸鼹鼠自然感染多瘤病毒的调查为实验用裸鼹鼠微生物标准的制定提供了参考。

Objective To establish a fluorescence quantitative PCR assay for polyomavirus and to apply this technique in the investigation of its infection rate in naked mole rats. Methods To compare the nucleic acid sequence of murine polyomavirus（ Genbank： NC_001515） in NCBI and design primers and probes in its conserved region. To establish a fluorescence quantitavive PCR method for polyomavirus and evaluate the sensitivity and specificity of the method. To infect nine one-day old KM strain suckling mice,and to collect samples of the heart,liver,spleen,lung,kidney,brain,thymus,cecal contents and blood at 21 days after infection. The efficacy of the method was validated by detecting the virus in organs. 62 cecal samples from naked mole rats were tested by the established assay. Results There was obvious fluorescence signal when polyomavirus was used as the template and no fluorescence signal when simian virus 40,murine K virus,MVM and H-1 were used as templates. The detection limit of the assay was 100 copies/μL. Polyomavirus DNA wasdetected in the heart,liver,spleen,lung,kidney and cecal contents of the mice which were inoculated with polyomavirus.The polyomavirus DNA content was highest in the lung tissue. There was no detectable polyomavirus DNA in the brain,thymus and blood of the infected mice. Sixty-two cecal contents of naked mole rats were tested for polyomavirus and the results were negative. Conclusions The fluorescence quantitative PCR assay for polyomavirus established in this study can effectively detect polyomavirus DNA in animal tissues. The results of investigation of the natural infected polyomavirus of naked mole rats provide a reference for the formulation of microbiological criteria for experimental naked mole rats.