感染性疾病RNA病毒性病原体生物信息学检测方法的建立

目的以艾滋病病毒(HIV)为研究对象,建立感染性疾病RNA病毒性病原体的生物信息学检测方法。方法应用DNase-非序列依赖的单引物扩增技术,将艾滋病患者血清过滤后,经DNase处理去除血清中的内源DNA,提取血清病毒RNA,反转录合成病毒RNA的双链cDNA,TaqⅠ酶切双链cDNA,加接头分子,并以接头分子为引物非特异扩增病原基因;PCR产物克隆后,酶切鉴定并测序,经GenBankBLAST软件进行序列分析。结果非序列依赖的单引物扩增HIV患者血清中外源基因得到多个DNA片段;重组克隆质粒经EcoRⅠ和HindⅢ双酶切,可见插入片段存在;将所有PCR产物测序,序列与已知病原基因序列一致。结论应用DNase处理及非序列依赖的单引物扩增方法可以检测RNA病毒性病原体。

Development of A Bioinformatic Method for Determination of RNA Viral Pathogen of Infectious Diseases

Objective To develop a bioinformatic method for determination of RNA viral pathogen of infectious diseases using HIV as subject. Methods The sera of patients with AIDS were filtered then treated with DNase to remove endogenous DNA by DNase sequence-independent single primer amplification. Viral RNA was extracted from the sera and reversely transcribed to doublestranded cDNA which was digested with Taq Ⅰ and added with adapter molecule as a primer for non-specific amplification of pathogenic gene by PCR. The PCR products were cloned and identified by restriction analysis, and their sequences were compared with those reported in GenBank by using BLAST software. Results Several DNA fragments were obtained from sera of patients with AIDS by DNase sequence-independent single primer amplification. The inserted gene fragment was observed in constructed recombinant cloning vector after digestion with EcoRⅠ and Hind Ⅲ. All the sequences of PCR products were consistent with those of known pathogenic genes. Conclusion RNA viral pathogen may be detected by DNase treatment and DNase sequence-independent single primer amplification.