Development and validation of a real-time PCR for detection of pathogenic leptospira species in clinical materials.
Development and validation of a real-time PCR for detection of pathogenic leptospira species in clinical materials.
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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。
DOI：
10.1371/journal.pone.0007093
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发表时间：
2009-09-18
期刊：
影响因子：
3.7
通讯作者：
Hartskeerl RA
中科院分区：
文献类型：
--
作者：
Ahmed A;Engelberts MF;Boer KR;Ahmed N;Hartskeerl RA
现有的血清学诊断不允许在疾病的早期急性期确认临床疑似钩端螺旋体病。几个传统的和实时PCR的钩端螺旋体病的早期诊断已被描述，但这些已不完全评估。我们开发了一种基于SYBR Green的靶向secY的实时PCR，并根据国际指南对其进行了验证。为了确定分析特异性，来自属于致病性、非致病性和中间型钩端螺旋体属的56株钩端螺旋体的DNA。以及46种其他微生物被包括在本研究中。所有致病性钩体均呈阳性反应。我们发现与腐生钩端螺旋体和其他微生物没有交叉反应，这意味着分析特异性高。PCR的分析灵敏度从培养的同源菌株M 20为1拷贝/反应，而异源菌株1342 K和Sarmin分别为1.2和1.5拷贝/反应。在加标的血清、血液和肾组织中，灵敏度对于M 20为10和20拷贝，对于1342 K为15和30拷贝，对于Sarmin为30和50拷贝。为了确定诊断的敏感性（DSe）和特异性（DSp），临床血液样本从26个实验室确诊和107个阴性疑似钩端螺旋体病患者作为一个前瞻性的连续队列。以培养物为金标准，我们发现DSe和DSp分别为100%和93%。所有8份PCR阳性样本的培养物均为阴性，随后发生血清转化，表明实际DSp较高。当使用培养和血清学作为金标准时，DSe较低（89%），而DSp较高（100%）。在发病后第一天（对于治疗很重要）4天内收集的样本中，DSe为100%。通过对20只确认阳性和20只阴性啮齿动物的肾脏样本进行盲法检测，测定的重现性和重复性均为100%。总之，我们已经描述了第一次的发展，一个强大的SYBR绿色实时PCR检测致病性钩端螺旋体结合其临床准确性的详细评估，从而提供了一种方法，钩端螺旋体病的早期诊断具有良好的定义令人满意的性能。
Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
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影响因子：
3.2
作者：
JOHNSON, RC;HARRIS, VG
通讯作者：
HARRIS, VG
影响因子：
3.1
作者：
Haake, DA;Chao, G;Bolin, CA
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Bolin, CA
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作者：
BAL, AE;GRAVEKAMP, C;TERPSTRA, WJ
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TERPSTRA, WJ
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11.8
作者：
Gouvela, Edilane L.;Metcalfe, John;Ko, Albert I.
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Ko, Albert I.
影响因子：
3
作者：
do Oliveira, MAA;Caballero, OL;Koury, MC
通讯作者：
Koury, MC