Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay
Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay
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DOI：
10.1128/jcm.43.4.1851-1857.2005
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发表时间：
2005-04-01
影响因子：
9.4
通讯作者：
Ou, CY
中科院分区：
文献类型：
--
作者：
Luo, W;Yang, H;Ou, CY
干血斑（DBS）是用于人类免疫缺陷病毒（HIV）研究的各种流行病学、血清学和分子检测的血液样本采集、运输和储存的公认方法。这对于在偏远地区受影响的个人进行母婴传播研究尤为重要。我们开发了一种实时PCR方法来检测干血斑中的HIV-1 DNA。将细胞基因RNase P与HIV-1 DNA在同一试管中共扩增，以监测DNA提取效率和总体测定性能。我们的检测试剂盒是一种单管、单步封闭系统检测试剂盒，使用dUTP/尿嘧啶DNA糖苷酶抗PCR污染对照。HIV-1引物和探针均来源于长末端重复序列的保守区域。通过降低正向和反向引物浓度来减弱RNase P的检测，使得其扩增不会压倒HIV-1扩增，并且还将提供分离的DBS DNA的质量的半定量测量。我们检查了103名HIV-1血清阳性和56名血清阴性的美国成年人，发现我们的检测方法的灵敏度为98.1%（95%置信区间[CI]，95.5%至100%），特异性为100%（95% CI，99%至100%）。阳性和阴性预测值分别为100%和96.6%。这种双重PCR检测可能有助于识别HIV-1感染者，特别是世界偏远地区血清阳性母亲所生的婴儿。
A dried blood spot (DBS) is a well-accepted means for the collection, transport, and storage of blood samples for various epidemiologic, serologic, and molecular assays for human immunodeficiency virus (HIV) studies. It is particularly important for mother-to-infant-transmission studies of affected individuals living in remote areas. We have developed a real-time PCR method to detect HIV type 1 (HIV-1) DNA in dried blood spots. A cellular gene, RNase P, was coamplified with the HIV-1 DNA in the same tube to monitor the DNA extraction efficiency and the overall assay performance. Our assay is a one-tube, single-step closed-system assay and uses a dUTP/uracil DNA glycosidase anti-PCR contamination control. The HIV-1 primers and probe were derived from a conserved region of the long terminal repeat. The detection of RNase P is attenuated by lowering the forward and reverse primer concentrations so that its amplification will not overwhelm the HIV-1 amplification and yet will provide a semiquantitative measurement of the quality of the isolated DBS DNA. We examined 103 HIV-1-seropositive and 56 seronegative U.S. adults and found that our assay has a sensitivity of 98.1% (95% confidence interval [CI], 95.5% to 100%) and specificity of 100% (95% CI, 99% to 100%). The positive and negative predictive values are 100% and 96.6%, respectively. This duplex PCR assay may be useful in identifying HIV-1-infected persons, particularly infants born to seropositive mothers in remote areas of the world.