Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis
Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis
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DOI：
10.1128/jcm.42.3.1207-1213.2004
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发表时间：
2004-03-01
影响因子：
9.4
通讯作者：
Pauli, G
中科院分区：
文献类型：
--
作者：
Nitsche, A;Ellerbrok, H;Pauli, G
虽然天花病毒在20世纪70年代被世界卫生组织的疫苗接种计划根除，但在生物恐怖袭击中可能故意释放天花病毒的背景下，天花感染的诊断引起了极大的兴趣。显然，需要快速和可靠的诊断工具来检测天花病毒并将其与具有相同形态特征的正痘病毒（包括牛痘病毒）区分开。用于病毒感染的临床诊断的实时PCR的出现促进了以快速、安全和精确的方式检测微量的病毒核酸，包括可靠地定量和基因分型靶标的选择。在这项研究中，一套完整的四杂交探针为基础的实时PCR检测正痘病毒DNA的特异性检测。PCR后的解链分析能够通过PCR产物的特征性解链温度鉴定天花病毒，从而将天花病毒与其他正痘病毒区分开来。此外，提出了用于天花病毒DNA的特异性扩增的测定。所有检测可在同一台循环仪中同时进行，PCR运行结果可在1小时内获得。对同一种微生物应用一种以上的检测方法可显著提高诊断可靠性，降低因未知序列变异而导致假阴性结果的风险。总之，所提出的测定将提高正痘病毒诊断和天花病毒鉴定的速度和可靠性。
Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.