Improved PCR Methods for Detection of African Rabies and Rabies- Related Lyssaviruses
Improved PCR Methods for Detection of African Rabies and Rabies- Related Lyssaviruses
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DOI：
10.1128/jcm.01256-10
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发表时间：
2010-11-01
影响因子：
9.4
通讯作者：
Markotter, Wanda
中科院分区：
文献类型：
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作者：
Coertse, Jessica;Weyer, Jacqueline;Markotter, Wanda
目前已知有11种不同的狂犬病毒属，其中4种发生在非洲大陆。这些病毒引起狂犬病，发展中国家的负担最高，那里通常没有常规的实验室诊断。从流行病学和控制的角度来看，诊断方法有必要检测世界不同地区存在的狂犬病病毒的多样性。一个出版的和广泛使用的半巢式逆转录-PCR（hnRT-PCR）的能力进行了评估，以检测一组不同的非洲狂犬病毒。由于该试验的局限性，开发了替代hnRT-PCR。发现新的检测方法在检测各种临床标本中的非洲狂犬病病毒RNA时准确且灵敏。该测定进一步适应于实时PCR平台，以允许快速、一步、定量和单探针检测，并包括用于验证样品制备的内部对照。实时PCR检测的检测限为每个反应10个RNA拷贝，检测间和检测内变异性低于4%。随后，在证明实用性方面，两种检测方法均成功应用于人类死前狂犬病诊断。我们相信，定量实时PCR检测可以找到应用程序作为一个常规的确认测试狂犬病诊断在未来，它将作为一个有价值的研究工具，在非洲狂犬病病毒的生物学。或者，hnRT-PCR测定可用于无法获得昂贵的实时PCR设备的实验室，用于狂犬病毒属病毒的灵敏诊断。
Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter-and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses.