小鼠腺病毒荧光定量PCR方法的建立及初步应用

Establishment and Preliminary Application of a qPCR Detection Method for Mouse Adenovirus

目的基于TaqMan建立小鼠腺病毒(MAdV)特异的荧光定量PCR检测方法,用于调查长爪沙鼠和小鼠等实验动物中MAdV感染情况。方法从NCBI下载小鼠腺病毒(MAdV-A和MAdV-3)基因组序列,比对分析后选择保守性较好的基因设计引物和探针。筛选最佳引物对和探针,对其特异性、敏感性、重复性和稳定性进行验证,运用所建立的方法对长爪沙鼠和野鼠的共41份样本进行检测。结果经特异性和灵敏度鉴定,在设计的3对引物探针中确定一对最佳引物探针MAD-3,可区分小鼠腺病毒与猴腺病毒、禽腺病毒和树鼩腺病毒等24种病原,MAdV质粒DNA最低检测限为4.5 copies/reaction;MAdV纯病毒液和病毒/组织模拟样本的最低检测限均为440 copies/reaction。结论建立的Taq Man荧光定量PCR检测方法特异、灵敏和稳定,可用于大鼠、小鼠、长爪沙鼠等实验动物及鼠源性生物制品MAdV的检测。

Objective Establish a specific qPCR detectionmethod based on TaqMan probe to investigate mouse adenovirus （MAdV） infection in gerbils, mice and other laboratory animals. Method Genomic sequences of mouse adenoviruses （MAdV-A and MAdV-3） were downloaded and primers and probes were designed at the best conservative genes which were selected after comparative analysis. Screened and tested the specificity, sensibility, repeatability and stability of the best primers and probe, utilized this q-PCR method to detect 41 samples of gerbils and voles. Result MAdV-3 was chosen as the best from three pairs of primers and probes, which could distinguish MAdV, SAdV, FAV and TAV and other 24 viruses, the minimum detectable amount of MAdV plasmid DNA template is 4.5copies/reaction; the minimum detectable amount of MAdV pure virus and virus/tissue simulate sample DNA template both are 440copies/reaction. Conclusion The TaqMan qPCR detection method established in this manuscript has good specificity, sensitivity, repeatability and stability, which might be used to detect MAdV in laboratory animals and relevant biological products.