Development of a real-time PCR assay for the diagnosis of scrub typhus cases in India and evidence of the prevalence of new genotype of O-tsutsugamushi
Development of a real-time PCR assay for the diagnosis of scrub typhus cases in India and evidence of the prevalence of new genotype of O-tsutsugamushi
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DOI：
10.1016/j.actatropica.2007.07.013
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发表时间：
2007-10-01
期刊：
影响因子：
2.7
通讯作者：
Batra, Harsh Vardhan
中科院分区：
文献类型：
--
作者：
Bakshi, Diprabhanu;Singhal, Pradeep;Batra, Harsh Vardhan
应用针对东方体主要外膜抗原基因56 kDa截短片段设计的实时荧光定量聚合酶链式反应技术，从疑似患者的血液或血清中检测斑疹伤寒。对66份疑似斑疹伤寒和/或Weil-Felix试验阳性(>；=1：160)的发热患者的血液和/或血清样本进行了检测。对扩增产物进行终点熔融曲线分析和测序，证实了该方法的特异性。对样本进行套式聚合酶链式反应，以确定钩端螺旋体的血清型。在实时荧光定量聚合酶链式反应中，73%的可疑样品获得了强阳性荧光。一些阳性样本的血清型特异性聚合酶链式反应扩增表明是黑木型，而其余的对这项检测没有反应。对聚合酶链式反应扩增片段的序列分析表明，该区域存在以前未被描述的新的恙虫病原虫类型。当单独使用或与Weil-Felix试验一起使用时，这种一步实时聚合酶链式反应可用于检测和确认斑疹伤寒，这在大多数发展中国家仍然是唯一可用的斑疹伤寒检测方法。需要进行详细的研究，以进一步评估其作为诊断斑疹伤寒的特定分子工具的适宜性，并描绘这些地区的流行毒株类型，以便清楚地了解这种新出现的传染病。(C)2007 Elsevier B.V.保留所有权利。
A qualitative syber green real-time PCR with primers designed for a truncated portion of the 56 kDa major outer membrane antigen gene of Orientia tsutsugamushi was used to diagnose scrub typhus from the blood or serum of suspected patients. Sixty-six blood and/or sera samples from fever cases, either with high index of suspicion for scrub typhus and/or positive by Weil-Felix test (>= 1: 160), were tested with the PCR. Specificity of the PCR was confirmed by end point melt curve analysis and sequencing of the amplicons. A nested PCR for determination of the serotypes of O. tsutsugamushi was performed on to the samples. In real-time PCR strong positive fluorescence was obtained in 73% of the suspected samples. Serotype-specific PCR amplification of some of the positive samples was indicative of the Kuroki type whereas the rest were non-responsive to this test. Sequence analyses of PCR amplicons indicated the presence of new, previously undescribed type of O. tsutsugamushi in this region. This one-step real-time PCR can be used for the detection and confirmation of scrub typhus, when used independently or in conjunction with, the Weil-Felix test, which is still the only available detection test for scrub typhus in most parts of the developing world. Elaborate studies need to be taken up to further evaluate its suitability as specific molecular tool for the diagnosis of scrub typhus and to delineate the prevalent strain types in these regions for a clear epidemiological understanding of this emerging infectious disease. (C) 2007 Elsevier B.V. All rights reserved.