Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Thailand
Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Thailand
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DOI：
10.4269/ajtmh.2005.73.850
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发表时间：
2005-11-01
影响因子：
3.3
通讯作者：
Rosenblatt, J
中科院分区：
文献类型：
--
作者：
Swan, H;Sloan, L;Rosenblatt, J
我们比较了297例来自泰国的疟疾患者的诊断，通过实时聚合酶链反应（PCR）检测使用LightCycler与传统的显微镜使用Giemsa染色的厚和薄的血膜。PCR检测可在1小时内完成，并有可能通过使用熔解温度曲线分析在单个反应中检测和鉴定4种疟原虫（然而，我们在本研究中未检测到卵形疟原虫）。在IsoCode STIX上采集、储存和运输血液，这为DNA的存档和快速简单提取提供了稳定的基质。使用对应于18 S核糖体RNA的属特异性引物组来扩增靶序列。荧光共振能量技术杂交探针被设计用于恶性疟原虫在含有碱基对错配的区域上，这允许区分其他疟原虫物种。297份患者标本中有282份（95%）的PCR结果与显微镜结果相关。其中绝大多数为间日疟原虫和恶性疟原虫引起的单种感染（150例），沿着三日疟原虫5例，恶性疟原虫和间日疟原虫混合感染2例，阴性标本5例。阴性显微镜标本经PCR检测均为阳性（检测任何疟原虫的特异性为100%）。15个不一致结果无法解决，但鉴于显微镜检查的主观性和PCR的分析客观性，PCR结果可能是正确的。本研究无法确定PCR方法检测混合感染或检测卵形疟原虫的能力。在初始设备成本的限制下，这种实时PCR检测是一种快速、准确和有效的疟疾特异性诊断方法。它可以应用于临床实验室，以及流行病学研究和抗疟疗效试验。
We compared the diagnosis of malaria in 297 patients from Thailand by a real-time polymerase chain reaction (PCR) assay using the LightCycler with conventional microscopy using Giemsa-stained thick and thin blood films. The PCR assay can be completed in one hour and has the potential to detect and identify four species of Plasmodium in a single reaction by use of melting temperature curve analysis (however, we did not detect Plasmodium ovale in this study). Blood was collected, stored, and transported on IsoCode STIX, which provide a stable matrix for the archiving and rapid simple extraction of DNA. A genus-specific primer set corresponding to the 18S ribosomal RNA was used to amplify the target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing basepair mismatches, which allowed differentiation of the other Plasmodium species. The PCR results correlated with the microscopic results in 282 (95%) of 297 patient specimens. Most of these were single-species infections caused by P. vivax (150) and P. falciparum (120), along with 5 P. malariae, 2 mixed infections (P. falciparum and P. vivax), and 5 negative specimens. No negative microscopy specimens were positive by PCR (100% specificity for detection of any Plasmodium). The 15 discrepant results could not be resolved, but given the subjective nature of microscopy and the analytical objectivity of the PCR, the PCR results may be correct. The ability of the PCR method to detect mixed infections or to detect P. ovale could not be determined in this study. Within the limitations of initial equipment costs, this real-time PCR assay is a rapid, accurate, and efficient method for the specific diagnosis of malaria. It may have application in clinical laboratories, as well as in epidemiologic studies and antimalarial efficacy trials.