Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies (CRREM), St. Orsola Malpighi University Hospital, University of Bologna, Via G. Massarenti 9, 40138 Bologna, Italy.
J Clin Virol. 2011 Mar;50(3):221-3. doi: 10.1016/j.jcv.2010.11.008. Epub 2010 Dec 14.
Usutu virus (USUV), a flavivirus that belongs to the Japanese encephalitis virus (JEV) family, has recently emerged as a human pathogen, necessitating new diagnostic tools.
The development and assessment of a real-time RT-PCR assay to detect USUV in human samples.
Based on USUV genomic sequences from GenBank, USUV-specific primers and probes that target the NS5 gene were designed. The sensitivity was evaluated in a 10-fold dilution series of plasmid that contained the amplicon and in a dilution series of a quantified human USUV isolate. The specificity was determined by testing various concentrations of related ArBo viruses, including flaviviruses and phleboviruses. Human RNAse P was also amplified in the assay. One hundred four human specimens from patients who suffered from viral meningoencephalitis were evaluated.
The real-time RT-PCR assay had a sensitivity of 50 genomic copies per reaction (corresponding to 2200 copies/ml) and 1 PFU/ml of USUV isolate. USUV isolates from Austria were identified with identical efficiency, and no ArBo viruses, other than USUV, were detected. USUV was also identified in 3 cerebrospinal fluid samples. All human samples were positive for RNAse P.
This PCR assay is recommended for all cases in which a rapid and clinically accurate diagnosis of human USUV infection is required.
乌苏图病毒（USUV）是一种黄病毒，属于日本脑炎病毒（JEV）家族，最近已成为人类病原体，需要新的诊断工具。
开发和评估一种实时 RT-PCR 检测方法，用于检测人体样本中的 USUV。
基于来自 GenBank 的 USUV 基因组序列，设计了针对 NS5 基因的 USUV 特异性引物和探针。通过对包含扩增子的质粒进行 10 倍稀释系列和定量人 USUV 分离物的稀释系列来评估敏感性。通过测试各种浓度的相关 ArBo 病毒，包括黄病毒和节肢动物病毒，来确定特异性。该检测还扩增了人 RNAse P。评估了来自患有病毒性脑膜脑炎的患者的 104 个人体标本。
实时 RT-PCR 检测方法的灵敏度为 50 个基因组拷贝/反应（相当于 2200 拷贝/ml）和 1 PFU/ml 的 USUV 分离物。从奥地利分离的 USUV 具有相同的鉴定效率，并且未检测到除 USUV 以外的 ArBo 病毒。还在 3 份脑脊液样本中鉴定出 USUV。所有人类样本均对 RNAse P 呈阳性。
建议在需要快速和临床准确诊断人类 USUV 感染的所有情况下使用此 PCR 检测方法。
