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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 To establish a simple, rapid and effective method for the detection of Orientia tsutsugamushi using ring-mediated isothermal amplification (LAMP). Methods LAMP DESIGNER software was used to design universal detection primers for TSA56 gene of Orientia tsutsugamushi, and the detection method of LAMP for Orientia tsutsugamushi was established. The detection specificity and sensitivity were compared with Realtime PCR and normal PCR methods. Meanwhile, the detection rate of 16 clinically diagnosed cases of Orientia tsutsugamushi infection and 20 healthy people were compared. Results The results showed that there was no cross-reactivity with the other pathogens. The detection sensitivity of the LAMP method was 1.0 × 102 copies / μl, which was the sensitivity of the general PCR method (1.0 × 103 copies / μl). 16 cases of Oriental tsutsugamushi infection or blood samples of esophageal samples or esophageal samples were detected, the positive number of positive LAMP detection method and Real-time PCR (16/16), the detection rate was 100%, higher than the ordinary PCR method (62.5 %, 10/16). Three methods to detect healthy samples were negative. Conclusion This study successfully established a simple, specific and sensitive method for detecting LAMP of Orientia tsutsugamushi, which provided a new method for the diagnosis of Orientia tsutsugamushi.