Department of Clinical Microbiology, Infectious Diseases, Umeå University, SE-901 85 Umeå, Sweden.
J Clin Virol. 2013 Apr;56(4):280-5. doi: 10.1016/j.jcv.2012.12.001. Epub 2013 Jan 3.
Hantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics.
This paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses.
Primers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments.
By using SYBR-green-based QRT-PCR 100-1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV.
This novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species.
汉坦病毒在世界大部分地区流行，每年在欧亚大陆和美洲造成成千上万例肾综合征出血热（HFRS）和汉坦病毒心肺综合征（HCPS）。它们是动物源性病毒，最常通过气溶胶化啮齿动物粪便传播给人类。新的汉坦病毒经常在以前未知的宿主物种和地理区域中被发现。因此，有必要改进汉坦病毒的诊断方法。
本文描述了一种快速而稳健的定量实时 PCR（QRT-PCR）检测方法的设计和评估，该方法能够检测广泛的汉坦病毒。
从不同汉坦病毒 L 片段的多个序列比对中确定的保守区域设计了具有检测不同汉坦病毒潜力的引物。
通过使用 SYBR-绿色 QRT-PCR，能够检测到体外产生的 RNA 的 100-1000 个目标分子，以及来自不同汉坦病毒分支和世界不同地区的少于 100 个拷贝的汉坦病毒 RNA。当使用该检测方法对急性 HFRS 患者的临床样本进行检测时，在所有之前为阳性的样本中都证实了普马拉病毒（PUUV）RNA。值得注意的是，广谱反应的 L 片段 QRT-PCR 还在 HFRS 患者样本中检测到了病毒 RNA，而这些样本之前用针对 PUUV S 片段的 QRT-PCR 检测为阴性。
该新型检测方法为来自不同分支和地区的汉坦病毒的诊断提供了一个强大的工具，也可能对在啮齿动物或食虫动物物种中寻找新汉坦病毒的调查有用。
