Real-time PCR for detection of Trypanosoma brucei in human blood samples
Real-time PCR for detection of Trypanosoma brucei in human blood samples
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DOI：
10.1016/j.diagmicrobio.2004.07.001
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发表时间：
2004-11-01
影响因子：
2.9
通讯作者：
Steverding, D
中科院分区：
文献类型：
--
作者：
Becker, S;Franco, JR;Steverding, D
我们建立了一种实时荧光PCR方法检测人体血液中的布氏锥虫DNA。用新设计的针对布氏锥虫177 bp重复序列卫星DNA的引物进行PCR，并用Sybr绿色监测扩增子积累。使用Chelex 100（R）树脂对收集在Whatman FTA（R）卡上的血液样品进行DNA纯化，并且如通过实时PCR所揭示的，显示出是一种简单且定量的方法。检测限为100锥虫每毫升血液，相当于0.1基因组当量的分析灵敏度。在昏睡病患者的所有血液样品中检测到锥虫DNA，此外，在所有测定中通过解离分析确认扩增子的身份。尽管来自血液样品的模板DNA的扩增效率显著低于基因组DNA，但所有测定之间的相似效率确保了定量结果。未感染个体的样品未获得扩增子产物。结果表明，实时荧光PCR检测方法是一种快速，灵敏的方法，适用于检测人体血液样本中的布氏锥虫在常规的临床实验室实践。(C)2004年爱思唯尔公司All rights reserved.
We have developed a real-time PCR assay for detection of Trypanosoma brucei DNA in human blood samples. The PCR was conducted with newly designed primers targeting the 177-bp repeat satellite DNA in T brucei and with Sybr Green to monitor the amplicon accumulation. DNA purification using Chelex 100(R) resin was performed on blood samples collected on Whatman FTA(R) cards and was shown to be a simple and quantitative method as revealed by real-time PCR. The detection limit of the assay was 100 trypanosomes per mL blood, corresponding to an analytical sensitivity of 0.1 genome equivalents. Trypanosome DNA was detected in all blood samples from sleeping sickness patients and, furthermore, the identity of the amplicon was confirmed in all assays by dissociation analysis. Although template DNA from blood samples was amplified with significantly lower efficiency than genomic DNA, similar efficiency between all assays ensured quantitative results. No amplicon product was obtained with samples from uninfected individuals. The results indicate that the real-time PCR assay described is a rapid and sensitive method suitable for the detection of T brucei in human blood samples in routine clinical laboratory practice. (C) 2004 Elsevier Inc. All rights reserved.