用两步MS-PCR结合ELISA检测HIV-1逆转录酶基因的耐药性突变

中国生物工程杂志

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用两步MS-PCR结合ELISA检测HIV-1逆转录酶基因的耐药性突变

何红秋程绍辉刘斌陈慰祖王存新

北京工业大学生命科学与生物工程学院北京工业大学生命科学与生物工程学院北京工业大学生命科学与生物工程学院北京工业大学生命科学与生物工程学院北京工业大学生命科学与生物工程学院

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

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摘要： 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。

Abstract: 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. In this work, 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. We designed a longer mutant type primer, 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. We suggest that it will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

收稿日期: 2006-04-20 出版日期: 2006-08-25

通讯作者: 何红秋

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引用本文:

何红秋,程绍辉,刘斌,陈慰祖,王存新. 用两步MS-PCR结合ELISA检测HIV-1逆转录酶基因的耐药性突变[J]. 中国生物工程杂志, .

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

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I08/42

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