树鼩腺病毒（TAV）PCR方法的建立及初步应用

摘要

Objective To establish and apply an effective PCR assay for detection of the Tupaia ( tree shrew) adenovirus ( TAV) .Methods According to NCBI Genbank, TAV genome DNA from 19418 to 19917 were synthetized and inserted into a plasmid as positive standards.One pair of primers was designed based on this sequence.Sixty blood samples and fifty-six stool samples from tree shrew were detected with this PCR assay.Results A PCR method for detection of TAV was successfully established, with a high specificity and the sensitivity was 13.5 ×10 -7μg/mL.The PCR results of testing sixty tree shrew blood DNA samples were negative.24 positive cases were tested among 56 stool DNA samples.Sequencing of the samples confirmed a 42.9%infection rate of TAV in tree shrew stool samples, well consistent with the PCR results.Conclusions The PCR method for detecting TAV established in this study has good specificity and high sensitivity, therefore, can be used in conventional detection of tree shrew adenovirus.%目的：建立树鼩腺病毒（ TAV） PCR检测方法，并进行初步应用。方法根据NCBI公布的TAV基因组序列，选择19418-19917区域人工合成一段DNA序列并转入质粒中，作为阳性标准质粒。设计1对特异性引物建立TAV PCR检测方法，考察其特异性和灵敏度。应用该PCR方法对60份树鼩血DNA及56份树鼩粪便DNA进行检测。结果所建立的PCR检测方法经特异性和灵敏度测定，最低可检测13.5 x10－7μg／mL，与其他腺病毒无交叉，特异性好，灵敏度较高。60份树鼩血DNA检测均为阴性，56份树鼩粪便DNA检测有24份阳性，通过测序证实树鼩粪便中TAV感染率为42.9％，与扩增结果一致。结论建立的树鼩腺病毒PCR检测方法特异性强，灵敏度高，可用于树鼩腺病毒的常规检测中。