磁珠分离DNA技术检测HIV-1逆转录酶基因耐药性突变

Detection of Drug-resistant Mutations in HIV-1 RT Gene Using Magnetic Beads Associated DNA Isolation Technology

HIV-1逆转录酶基因突变导致的耐药性严重影响了药物对病人的治疗效果,耐药性突变的检测对于指导合理用药具有重要意义。发展了一种检测HIV-1逆转录酶基因耐药性突变的新方法,在两步MS-PCR方法的基础上,引入磁珠分离DNA技术并结合酶联免疫吸附检测方法(ELISA)检测逆转录酶基因耐药性突变T215F和Y181C。结果显示:MS-PCR结合磁珠分离技术和ELISA具有较高的灵敏度和特异性,阳性/阴性比值(P/N值)达到要求,突变型模板检测限为5%左右,耗时短且能进行高通量检测。无论在HIV-1耐药性突变还是其它的点突变检测中,该方法都具有较好的临床应用前景。

In recent years, the emergence of drug-resistant RT gene mutations in HIV-1 strains has become the major reason for AIDS treatment failure. The detection of drug-resistant mutations is important for the treatment choice. A novel assay for the detection of HIV-1 RT drug-resistant mutations was developed. Drugresistant and wild strains of HIV-1 B subtype were investigated based on Two-Step MS-PCR. Magnetic beads associated DNA isolation technology and ELISA were employed in this assay to detect point mutations including Y181C and T215F. As a result, this novel assay gave a good P/N ratio and as less as about 5% of mutant type template could be detected, indicating that this assay is highly sensitive and specific. The assay can be completed in relatively less time consumption and applied in high-throughput screening. The method has great potential for using in clinic assay for the detection of HIV-1 drug-resistant mutations as well as other point mutations.