Myint Lay, Ariyoshi Koya, Yan Hua, Frater Alexander J, Auwanit Wattana, Pathipvanith Panita, Yamada Kaneo, Matsuda Masakazu, Chiba Tomoko, Fujita Kazunori, McClure Myra, Weber Jonathan N, Sugiura Wataru
AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
Antimicrob Agents Chemother. 2002 Dec;46(12):3861-8. doi: 10.1128/AAC.46.12.3861-3868.2002.
A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.
基于诱变分离聚合酶链反应（MS-PCR）技术开发了一种快速的齐多夫定（ZDV）耐药基因型检测方法，用于检测CRF01_AE（E亚型）中的两种ZDV耐药突变，即M41L和K70R。终点稀释分析表明，新构建的MS-PCR检测方法能够成功检测到三至九个拷贝的1型人类免疫缺陷病毒模板RNA。针对不同比例的野生型和突变型模板混合物进行的检测表明，该检测方法至少能够检测到10%的次要群体。采用新构建的MS-PCR检测方法和直接核苷酸测序对51份E亚型临床样本进行了分析。两种检测方法在第41和70密码子处的一致性分别为92%和100%。MS-PCR检测方法是一种快速、简单且廉价的检测方法，在检测包括次要群体在内的突变靶点方面具有高度敏感性。因此，它可作为发展中国家耐药突变流行病学监测的有力工具。
