用两步MS-PCR结合ELISA检测HIV-1逆转录酶基因的耐药性突变

高效抗逆转录病毒治疗法（HAART）的推广使用有效地抑制了HIV-1病毒的传播和艾滋病（AIDS）的发病率、死亡率。近年来，HIV-1逆转录酶基因突变所导致的耐药性成为了HAART的治疗失败的主要原因，耐药性突变的检测对于指导病人用药以及新药开发具有重要意义。本工作发展了一种检测HIV-1逆转录酶基因耐药性突变的新方法。采用两步MS-PCR方法检测HIV-1 B亚型野生型和耐药突变型逆转录酶基因突变，检测点包括M41L、K70R、K103N、Y181C和T215，并在两步MS-PCR基础上，设计比野生型引物长的突变型引物，结合微孔板杂交ELISA呈色技术检测各点突变。结果显示，简化的两步MS-PCR能保持灵敏度和特异性高的优点，ELISA的阳性结果与阴性结果的比值（P/N）达到要求，两步MS-PCR结合ELISA方法灵敏度高，操作简单、结果直观、成本低、相对耗时短且能进行高通量检测，其无论在HIV-1耐药性突变及其它的点突变检测中具有较好的临床应用前景。

Two-Step MS-PCR combined with ELISA Method for the detection of Drug Resistance Mutations in HIV-1 RT Gene

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.