Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA
Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA
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DOI：
10.1089/088922201750063160
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发表时间：
2001-02-01
影响因子：
1.5
通讯作者：
North, TW
中科院分区：
文献类型：
--
作者：
Leutenegger, CM;Higgins, J;North, TW
我们开发了一种基于实时PCR（TaqMan，Applied Biosystems，Foster City，CA）的快速且高度可重复的测定法，用于定量血浆样品中的猴免疫缺陷病毒（SIV）RNA。将该测定法与当前的支链DNA测定法（Bayer，Emeryville，CA）进行比较。在重叠的灵敏度范围内，用实时TaqMan PCR测定法获得的结果与用支链DNA测定法获得的结果相当（r = 0.9429，p < 0.05）。然而，实时TaqMan PCR检测能够检测低至50拷贝/ml的RNA，而支链DNA仅对1，500拷贝/ml的RNA敏感。因此，几只通过支链DNA检测为阴性的动物通过实时TaqMan PCR检测为阳性。支链DNA检测还记录了两次假阳性检测。假阴性和假阳性检测通过细胞培养分离和常规巢式RT-PCR证实。SIV TaqMan检测试剂可检测到多种具有相似扩增效率的野生型、克隆和重组SIV毒株，包括SIVmac 251、SIVmac 239、RT中含有184 V突变的SIVmac 239、SIV 1A 11、SIVmac 239 delta 3、SIVmac-M4和含有特定HIV-1基因（如逆转录酶（RT-SIV）或Env（SHIV-E））的嵌合体（SIV）。总之，实时SIV RNA定量的高灵敏度、增加的特异性、宽动态范围、简单性和再现性允许筛选大量样品，并且使得该方法特别适合于在疫苗和治疗研究期间测量病毒DNA和RNA水平。
We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by real-time TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.