Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis
Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis
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DOI：
10.1016/j.diagmicrobio2015.03.024
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发表时间：
2015-07-01
影响因子：
2.9
通讯作者：
Hoffmaster, Alex R.
中科院分区：
文献类型：
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作者：
Galloway, Renee L.;Hoffmaster, Alex R.
人类钩端螺旋体病的早期诊断对于开始适当的治疗至关重要;然而，金标准血清学试验直到症状发作后近一周才能检测到抗体。PCR在钩端螺旋体病的早期诊断中具有敏感性和特异性。之前，我们开发并验证了靶向lipL 32的TaqMan PCR测定。我们使用PerfeCfa（R）qPCR ToughMix（R），Low ROX（TM）（Quanta Biosciences，盖瑟斯堡，MD，美国）重新优化和验证了该测定。为了用新的混合物进行优化，与我们以前的测定相比，最终引物浓度从0.5 μ mol/L增加到0.9 μ mol/L，探针浓度从0.1 pmol/L增加到0.125 μ mol/L。这种新优化的检测方法降低了检测限，提高了诊断灵敏度。在这里，我们提出了改进的检测的性能数据，并描述了几个临床病例，最初是阴性的，但使用优化的检测阳性。爱思唯尔公司出版
Early diagnosis of leptospirosis in humans is critical with regard to initiation of appropriate treatment; however, the gold standard serological test cannot detect antibodies until nearly a week after symptom onset. PCR has been shown to be sensitive and specific in the early phase of leptospirosis. Previously, we developed and validated a TaqMan PCR assay targeting lipL32. We reoptimized and validated this assay using PerfeCfa (R) qPCRToughMix (R), Low ROX (TM) (Quanta Biosciences, Gaithersburg, MD, USA). For optimization with the new mix, the final primer concentrations were increased from 0.5 mu mol/L to 0.9 mu mol/L compared to our previous assay, and the probe concentration increased from 0.1 pmol/L to 0.125 mu mol/L. This newly optimized assay resulted in a lower limit of detection and increased diagnostic sensitivity. Here, we present the performance data of the improved assay and describe several clinical cases that were initially negative but tested positive using the optimized assay. Published by Elsevier Inc.