淋巴细胞脉络丛脑膜炎病毒实时 RT-PCR 检测方法的分析验证。

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay.

机构信息

Swiss National Reference Centre for Emerging Viral Diseases, University of Geneva Hospitals, Geneva, Switzerland.

出版信息

J Virol Methods. 2011 Oct;177(1):118-22. doi: 10.1016/j.jviromet.2011.06.018. Epub 2011 Jul 6.

DOI:10.1016/j.jviromet.2011.06.018

Abstract

Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology. A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory. A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10PFU/ml (Arm53b and WE54) and 1PFU/ml (Traub and E350). Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses. This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.

摘要

淋巴细胞性脉络丛脑膜炎病毒（Lymphocytic Choriomeningitis Virus，LCMV）是一种罕见的人类中枢神经系统疾病病原体。在不明病因的无菌性脑膜炎病例中，实时 RT-PCR 检测具有重要的筛查意义。本研究开发了一种基于 LCMV 病毒核蛋白（Nucleoprotein，NP）基因组序列检测的实时 RT-PCR 检测方法，用于评估瑞士一所大学医院实验室送检的脑脊液中是否存在 LCMV。使用包含 LCMV 分离株 Armstrong（Arm）53b 的 cDNA 的质粒进行 10 倍稀释系列检测，显示该方法具有高灵敏度，最低检测限为 ≤50 拷贝/反应。通过使用 4 种不同 LCMV 分离株（Arm53b、WE54、Traub 和 E350）在人 CSF 中的稀释系列检测，进一步证实了该方法的高灵敏度，观察到的检测限为 ≤10PFU/ml（Arm53b 和 WE54）和 1PFU/ml（Traub 和 E350）。对 130 例 CSF 的分析未发现急性感染病例。使用一种已发表的检测所有旧世界沙粒病毒的 PCR 检测方法对无阳性病例进行了确认。本研究验证了一种用于诊断 LCMV 感染的特异性和高灵敏度的实时 RT-PCR 检测方法。结果表明，LCMV 感染在瑞士西部的住院患者中极为罕见。