随机聚合酶链反应检测未知病毒方法学的建立

Establishment of a random PCR method for detecting unknown virus

目的探索并建立一种用于快速检测未知病毒的分子生物学方法。方法分别以乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)为假定的未知DNA、RNA病毒,验证随机聚合酶链反应(PCR)检测未知病毒的可行性。分离HBV、HCV的阳性血清,去除宿主DNA后,提取病毒核酸。锚定随机引物经Klenow酶处理(模板为DNA)或反转录酶作用(模板为RNA)退火至模板,随后用锚定特异引物对模板进行非特异性扩增。扩增产物经纯化后克隆、测序,最后与BLAST进行比对。结果经BLAST比对证实,插入序列中有HBV和HCV的基因组片段,在病毒为1×106拷贝/ml时,被检测克隆的阳性率约为15%。目前我们利用本法能达到的检测低限大致为1×104拷贝/ml。结论成功建立了一种基于随机PCR的未知病毒检测方法,其优点在于不依赖病毒的细胞培养及其核酸序列信息。这种方法的建立为快速检测不明原因疾病和新发传染病病原体提供了新的思路。

Objective To establish a molecular biology method for detecting sequences of unknown viruses. Methods The hepatitis B and hepatitis C viruses were used as hypothetically unrecognized DNA, RNA viruses respectively to test the efficacy of this random PCR- based method. Vires-containing serana was first filtered through a 0.22-gin disk filter followed by DNase I treatment to eliminate the host genomic DNA. After the 26mer 5＇ end anchored random primer was annealed to the extracted nucleic acids either by Klenow polymerase extension （DNA as template） or by reverse transcription （RNA as template）, the vires genomes were randomly amplified with the 20mer anchor primer. Purified PCR products were cloned, sequenced and subjected to BLAST search for further analysis. Results HBV and HCV genomes were anaplified by random PCR and their fragments could be detected in the picked clones. The detective rate was about 15% at 1 ×10^6 copies/ml viral load. Positive clones could still be obtained with as few as 1 ×10^4 copies/ml viral load. Conclusions This cultureindependent random PCR-based method can be used to investigate unknown viruses and will be useful for the early diagnosis of emerging infectious disease.