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首页> 外文期刊>Journal of Clinical Microbiology >Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC
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Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC

机译：人类免疫缺陷病毒1型B，C，CRF01-AE，CRF07-BC和CRF08-BC的简单亚型分析

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After the initial development of a human immunodeficiency virus type 1 (HIV-1) subtype-screening tool by nested multiplex PCR, we further improve it through the redesign of subtype-specific primers based on subtype signature pattern (SSP) analyses and optimization of the PCR conditions. Extracted RNA from plasma samples was used in reverse transcription and the cDNA products were added to the first round PCR, in which universal primers in the gag region were used to detect HIV-1 M group isolates. In the second round of PCR, three pairs of subtype-specific primers, detecting subtypes B, C, and CRF01-AE, were added in one tube. Subtype determination was based on the different size of PCR products on the agarose gel electrophoresis. An additional set of primers detecting only the prevalent recombinant strains CRF07-BC and CRF08-BC was used to discriminate CRF07- and CRF08-BC from pure subtype C. Testing for all kinds of HIV subtype reference strains indicated that this assay was applicable. A panel of 252 HIV-positive samples and 30 HIV-negative samples was further used to evaluate and validate this assay. Compared to the assay of sequence-based phylogenetic analysis, the newly developed assay has an adequate designated subtype sensitivity, 93.2% (69 of 74) for subtype B, 95.1% (117 of 123) for subtype C, 94.0% (47 of 50) for CRF01-AE, and 95.0% (115 of 121) for CRF07-BC and CRF08-BC. Most importantly, the intersubtype specificity of the assay was found to be 100%. The assay specificity was also found to be 100% when used to test 30 HIV-negative samples. The average reproducibility was 96.0% for subtype B, 96.7% for subtype C, and 95.0% for CRF01-AE. We have developed a simple, rapid, and low cost assay for screening subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC in China.
机译：在通过巢式多重PCR初步开发了人类免疫缺陷病毒1型（HIV-1）亚型筛选工具后，我们通过基于亚型特征码（SSP）分析和优化的亚型特异性引物的重新设计，进一步改进了它。 PCR条件。从血浆样品中提取RNA进行逆转录，并将cDNA产物添加到第一轮PCR中，其中 gag 区的通用引物用于检测HIV-1 M组分离株。在第二轮PCR中，将三对亚型特异性引物（检测B，C和CRF01-AE亚型）添加到一个试管中。亚型的确定基于琼脂糖凝胶电泳上PCR产物的不同大小。仅检测流行的重组菌株CRF07-BC和CRF08-BC的另一组引物用于将CRF07-和CRF08-BC与纯C亚型区分开。对所有HIV亚型参考菌株的测试表明该测定适用。进一步使用一组252个HIV阳性样品和30个HIV阴性样品来评估和验证该检测方法。与基于序列的系统发育分析测定相比，新开发的测定具有足够的指定亚型敏感性，对于B亚型为93.2％（74中的69），对于C亚型为95.1％（123中的117），94.0％（50中的47） CRF01-AE为9％），CRF07-BC和CRF08-BC为95.0％（121之115）。最重要的是，测定的亚型间特异性为100％。当用于测试30个HIV阴性样品时，测定特异性也为100％。 B型的平均重现性为96.0％，C型的平均重现性为96.7％，CRF01-AE为95.0％。我们开发了一种简单，快速且低成本的测定方法，用于筛选中国的B，C，CRF01-AE，CRF07-BC和CRF08-BC亚型。

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