Development of lipL32 real-time PCR combined with an internal and extraction control for pathogenic Leptospira detection.
Development of lipL32 real-time PCR combined with an internal and extraction control for pathogenic Leptospira detection.
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DOI：
10.1371/journal.pone.0241584
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发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Meijer MC
中科院分区：
文献类型：
--
作者：
Ahmed AA;Goris MGA;Meijer MC
至少有两种实时PCR用于钩端螺旋体病的早期诊断已被描述，评估和验证。然而，至少有一份其他报告建议对这些测定中使用的引物和探针进行调整和修饰，因为已经描述了其他钩端螺旋体属物种，并且使用的引物和探针与相应的靶序列严重错配。本研究基于lipL32基因建立了一种实时荧光PCR检测致病性钩端螺旋体的方法。本发明的方法包括基于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。优化杂交、退火和延伸温度（60°C）作为扩增反应中使用的DNA聚合酶的最佳温度。本方法具有较高的灵敏度和特异性，计算出的诊断灵敏度和特异性分别为93.0%和98.3%。此外，本方法包括能够容易地检测假阴性结果的内部对照和能够估计DNA提取效率的任选提取对照。
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.
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实时PCR的开发和验证，用于检测临床材料中致病性钩端螺旋体。
影响因子：
3.7
作者：
Ahmed A;Engelberts MF;Boer KR;Ahmed N;Hartskeerl RA
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Hartskeerl RA
影响因子：
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通讯作者：
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作者：
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通讯作者：
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影响因子：
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作者：
Takahashi, Y;Akase, K;Fukunaga, M
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