Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid
Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid
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DOI：
10.1128/jcm.41.7.3133-3141.2003
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发表时间：
2003-07-01
影响因子：
9.4
通讯作者：
Jerome, KR
中科院分区：
文献类型：
--
作者：
Lai, KKY;Cook, L;Jerome, KR
一个LightCycler和两个TaqMan实时PCR检测进行了评估，对较旧的PCR与液相杂交方法检测肠道病毒RNA在74例患者的样本。两步LightCycler和两步TaqMan格式彼此相关性良好（r（2）= 0.90），与液相杂交方法相比同样敏感，而一步重组Tth DNA聚合酶格式相当不敏感，仅在约一半的先前通过液相杂交呈阳性的患者样本中检测到肠道病毒RNA。利用10穆尔cDNA优化了两步TaqMan方法，并在我们的研究中评价的方法中证明了最高程度的分析灵敏度，能够可重复地定量低至510拷贝的肠道病毒RNA/ml脑脊液。这种新的检测方法可以在4小时内完成，劳动强度小得多，并且与鼻病毒的交叉反应性比液相杂交法低。因此，两步TaqMan测定法应证明可用于诊断肠病毒性脑膜炎与细菌性脑膜炎，从而导致及时和适当的临床管理，这可以为患者和医疗保健系统节省大量成本。
A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated well with each other (r(2) = 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant Tth DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 mul of cDNA and demonstrated the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.