Department of Medical Microbiology, OIE and National Collaborating Centre for Reference and Research on Leptospirosis, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
PLoS One. 2020 Nov 2;15(11):e0241584. doi: 10.1371/journal.pone.0241584. eCollection 2020.
At least two real-time PCRs for the early diagnosis of leptospirosis have been described, evaluated and validated. However, at least one other report suggested adaptation and modification of primers and probes used in these assays since additional Leptospira species have been described and the primers and probe in use possess a serious mismatch to corresponding target sequence. In this study we developed a real-time PCR for detection of pathogenic Leptospira based on the lipL32 gene. The present method consists of generic primers and probes based on target sequence of 10 pathogenic Leptospira species including Leptospira interrogans. The hybridization, annealing and extension temperature (60°C) were optimized as the optimal temperature of the DNA polymerase enzyme which is used in the amplification reaction. The present assay has a high analytical sensitivity and specificity; the calculated diagnostic sensitivity and specificity were 93.0% and 98.3% respectively. Moreover, the present method includes an internal control which enables easy detection of false negative results and an optional extraction control which enables the estimation of the DNA extraction efficiency.
至少已有两种实时 PCR 方法被描述、评估和验证用于钩端螺旋体病的早期诊断。然而，至少还有一份报告建议对这些检测中使用的引物和探针进行调整和修改，因为已经描述了其他的钩端螺旋体物种，并且使用的引物和探针与相应的靶序列存在严重不匹配。在这项研究中，我们基于 lipL32 基因开发了一种用于检测致病性钩端螺旋体的实时 PCR 方法。本方法由针对包括问号钩端螺旋体在内的 10 种致病性钩端螺旋体的靶序列的通用引物和探针组成。杂交、退火和延伸温度（60°C）是优化的，因为它是扩增反应中使用的 DNA 聚合酶的最佳温度。本检测方法具有很高的分析灵敏度和特异性；计算出的诊断灵敏度和特异性分别为 93.0%和 98.3%。此外，本方法包括内部对照，可轻松检测到假阴性结果，以及可选的提取对照，可估计 DNA 提取效率。
