恙虫东方体环介导恒温扩增技术检测方法的建立

An establishment of loop-mediated Isothermal amplification for the detection of Orientia tsutsugamushi

目的利用环介导恒温扩增技术(LAMP)建立一种简便、快速、有效的恙虫东方体检测方法。方法利用LAMP DESIGNER软件设计恙虫东方体TSA56基因通用检测引物,建立恙虫东方体LAMP检测方法。并与Realtime PCR和普通PCR方法进行检测特异性和灵敏度的比较。同时,对16例临床确诊的恙虫东方体感染病例以及20例健康人样本进行检出率的比较。结果本研究设计的恙虫东方体检测引物与其他病原体无交叉反应,所建立的恙虫东方体LAMP方法的检测灵敏度与Real-time PCR一致,为1.0×102 copies/μl,是普通PCR方法灵敏度(1.0×103copies/μl)的10倍。16例临床确诊恙虫东方体感染病例血液样本或焦痂样本的检测,LAMP方法阳性检出数与Real-time PCR一致(16/16),检出率为100%,高于普通PCR方法(62.5%,10/16)。三种方法检测健康人样本均为阴性。结论本研究成功建立了一种简便、特异、灵敏的恙虫东方体LAMP检测方法,为临床恙虫东方体的诊断提供了新方法。

Objective Toestablish a specific,sensitive and convenient method based on the loop-mediated isothermal amplification assay（LAMP） to detect Orientia tsutsugamushi（O. tsutsugamushi） in the early clinical diagnosis. Methods A set of LAMP primers in this study was designed with LAMP DESIGNER software for Orientia tsutsugamushi detection,and the sensitivity,specificity of the LAMP were compared with conventional PCR and Real-time PCR. The positive detection rates of 16 patient samples and 20 healthy controls were compared. Results No cross-reactions of LAMP primers were observed during the tests. The sensitivity of the established LAMP method was identical to the Real-time PCR method（1.0×102copies / μl）,and ten-fold higher than that of conventional PCR（1.0×103copies / μl）. In the clinical application of 16 clinical diagnosed blood or eschar samples,the LAMP method had a better positive diagnostic rate which were the same as Real-time PCR-100%（16 / 16）, and more sensitive than conventional PCR（62.5%,10 / 16）. All healthy control samples showed negative results with the three methods. Conclusion The LAMP method we established provided a more specific,sensitive,and convenient new approach for the clinical detection of O. tsutsugamushi in the early diagnosis.