Comparison of LigAmp and an ASPCR assay for detection and quantification of K103N-containing HIV variants
Comparison of LigAmp and an ASPCR assay for detection and quantification of K103N-containing HIV variants
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DOI：
10.1089/aid.2007.0224
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发表时间：
2008-04-01
影响因子：
1.5
通讯作者：
Eshleman, Susan H.
中科院分区：
文献类型：
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作者：
Church, Jessica D.;Towler, William I.;Eshleman, Susan H.
我们比较了Ligandine检测和ASPCR检测在临床试验中接受单剂量奈韦拉平的63名女性样本中检测和定量含K103 N的HIV变异体的能力。首先使用ViroSeq HIV Genotyping系统分析样本，并将ViroSeq PCR产物用作Ligands和ASPCR检测试剂的模板。两种试验均使用0.5% K103 N临界值检测K103 N。两种试验的K103 N百分比结果相似（R-2 = 0.92）。46份样本（73.0%）经两种检测均为K103 N阳性，13份样本（20.6%）经两种检测均为阴性。仅ASPCR阳性4例（6.3%）。无样本仅通过Ligandine呈阳性。更详细地分析了8个不一致样本。寡核苷酸结合位点附近的序列多态性提供了一个可能的解释，在八个样本中的四个不一致。K103 N百分比也通过使用组合扩增/测序方法（AmpliSeq）分析来自这8个样品中的每一个的40个HIV克隆来确定。通过克隆分析确定的K103 N百分比与8个样品中的5个样品的Ligandine结果一致，并且与8个样品中的3个样品的ASPCR结果一致。在分析的320个克隆中，我们在位置103处鉴定了8个不同的密码子（平均= 3.8个密码子/样品），其编码6个不同的氨基酸，说明了HIV中广泛的遗传多样性。需要进一步的研究来比较检测和定量临床样本中HIV耐药突变的检测方法的性能。
We compared the ability of the LigAmp assay and an ASPCR assay to detect and quantify K103N-containing HIV variants in samples from 63 women who received single- dose nevirapine in a clinical trial. Samples were first analyzed with the ViroSeq HIV Genotyping system, and ViroSeq PCR products were used as templates for the LigAmp and ASPCR assays. A cutoff of 0.5% K103N for detection of K103N was used for both assays. Results for the percentage K103N were similar for the two assays ( R-2 = 0.92). Forty- six samples ( 73.0%) were positive for K103N by both assays and 13 samples ( 20.6%) were negative by both assays. Four samples ( 6.3%) were positive by ASPCR only. No samples were positive by LigAmp only. Eight discordant samples were analyzed in more detail. Sequence polymorphisms near oligonucleotide binding sites provided a possible explanation for the discordance in four of eight samples. The percentage K103N was also determined by analyzing 40 HIV clones from each of these eight samples, using a combined amplification/sequencing method (AmpliSeq). The percentage K103N determined by clonal analysis was consistent with the LigAmp result for five of eight samples, and was consistent with the ASPCR result for three of eight samples. Among 320 clones analyzed, we identified eight different codons at position 103 ( mean = 3.8 codons/ sample), which encoded six different amino acids, illustrating the extensive genetic diversity in HIV. Further studies are needed to compare performance of assays for detection and quantification of HIV drug resistance mutations in clinical samples.