一种用于检测和定量脑脊液中 JC 多瘤病毒的环介导等温扩增检测方法：用于进行性多灶性白质脑病的诊断和临床管理的工具和技术。

A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

机构信息

Department of Virology 1, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, 162-8640, Japan.

Present Address: School of Tropical and Laboratory, Hainan Medical University, Hainan, 571199, China.

出版信息

Virol J. 2018 Aug 31;15(1):136. doi: 10.1186/s12985-018-1046-z.

DOI:10.1186/s12985-018-1046-z

PMID:30170628

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6119251/

Abstract

BACKGROUND

JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required.

METHODS

A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML.

RESULTS

The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 10 to 3.2 × 10 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay.

CONCLUSIONS

The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 10 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.

摘要

背景

JC 多瘤病毒（JCV）是进行性多灶性白质脑病（PML）的病原体，PML 是一种免疫抑制患者中枢神经系统的脱髓鞘疾病。PML 通常预后不良。检测和定量脑脊液（CSF）中的 JCV 基因组是诊断和管理 PML 的有效工具，需要进行适当的治疗干预。

方法

应用环介导等温扩增（LAMP）法对 JCV 进行定量检测。使用从疑似 PML 患者中收集的 153 份 CSF 标本，将 LAMP 检测与基于 TaqMan 的实时定量 PCR（qPCR）检测进行比较，评估其在 PML 诊断中的效果。

结果

LAMP 检测未与其他多瘤病毒质粒、病毒 DNA 和引起脑炎的病毒 RNA 发生交叉反应，并且在每个反应中检测到 1 个标准 DNA 拷贝。在 50 个 qPCR 阳性标本中，42 个标本（包含 JCV 基因组范围为 3.2×10 至 3.2×10 拷贝/反应）呈阳性反应，8 个标本（包含 0.9 至 19.9 拷贝/反应）呈阴性反应。此外，103 个 qPCR 阴性标本中有 3 个在 LAMP 检测中呈阳性反应。LAMP 检测的灵敏度、特异性、阳性预测值和阴性预测值分别为 84%（42/50）、97%（100/103）、93%（42/45）和 93%（100/108）。kappa 统计量为 0.83。对于 33 个拷贝数≥1 拷贝/反应的标本，LAMP 检测确定的 JCV 载量与 qPCR 检测确定的 JCV 载量呈强正相关（r=0.89）。此外，LAMP 检测与 qPCR 检测一样，可以对连续样本中的 JCV 基因组拷贝数进行监测。

结论

新开发的 LAMP 检测法对 JCV 具有高度特异性，可检测反应中每个样本 DNA 中包含 20 个或更多 JCV 基因组的 JCV 基因组，灵敏度为 100%（n=29），相当于 CSF 中的≥3×10 拷贝/ml。LAMP 检测法可用于诊断，并为临床环境中 PML 的评估和管理提供有价值的信息。