直接PCR法检测血液中伊氏锥虫感染

DETECTION OF KINETOPLAST DNA IN TRYPANOSOMA EVANSI BY DIRECT PCR FROM BLOOD SAMPLES

为建立伊氏锥虫动基体DNA(KinetoplastDNA,kDNA)的PCR扩增技术,血液直接PCR法检测伊氏锥虫的感染,本文以伊氏锥虫kDNA片段为靶扩增DNA设计引物,确定最适PCR反应条件,建立kDNA片段的PCR扩增技术,应用直接扩增缓冲液(Ampdirect)从感染小鼠的滤纸血标本直接PCR检测伊氏锥虫。结果发现,PCR扩增伊氏锥虫kDNA片段分子量为364bp,能检测的最小DNA量是0·06pg,与布氏锥虫、活动锥虫没有交叉反应。应用直接扩增缓冲液不需进行样本的DNA抽提,直接PCR能检测感染小鼠的早期滤纸血样本,敏感性高于镜检法。本实验建立的直接PCR法检测伊氏锥虫具有较高的敏感性和特异性,方法快捷,可进一步应用于伊氏锥虫病的早期诊断以及现场调查。

To establish a direct PCR method for the detection of Trypanosoma evansi from blood samples, primers were designed to amplify the DNA fragment of T. evansi kDNA under optimizing conditions. Ampdirect was applied to extract kinetoplast DNA from blood samples spotted on filter paper. The amplified fragment of DNA was 364 bp while there was no cross-reaction with T. brucei brucei and T. vivax, and it was capable to detect at least to 0.06 pg DNA of T. evansi. The direct PCR enabled to detect T. evansi infection 3 d after experimental inoculation, and with higher sensitivity than that of microscopic examination. The described direct PCR method is simple, rapid, highly sensitive and specific for T. evansi detection, and provide a valuable tool for early infection diagnosis and epidemiological survey of trypanosomosis.