粪便中隐孢子虫的提取与检测方法的建立

The establishment of extracting and detecting the cryptosporidium in human stool samples

目的建立粪便中隐孢子虫的提取与检测技术,分析腹泻病人粪便中隐孢子虫的感染情况,为该病的诊断、防治提供科学依据。方法选择经病原学检测已经确诊的5份隐孢子虫阳性和4份阴性粪便,分别采用FTA卡和商业化DNA抽提试剂盒抽提粪便样本中DNA,经荧光PCR法进行扩增,比较核酸提取效果;同时用直接涂片法、改良抗酸染色法及荧光PCR法对63例腹泻病人隐孢子虫感染情况进行粪样检测分析。结果应用FTA卡抽提DNA效果优于应用商业化DNA抽提试剂盒抽提DNA。同时对63份腹泻病人粪便运用直接涂片法、改良抗酸染色法及荧光PCR法都未检出隐孢子虫。结论应用FTA卡进行粪便样本中隐孢子虫DNA的抽提方法具有简单有效、快速灵敏、准确可靠等特点,可以应用于粪便中隐孢子虫的检测,同时可联合多种检测手段来提高腹泻病人原虫感染的检出率。

Objective To develop a kind of technique for extracting and detecting the cryptosporidium in human stool samples,and understand the infection in the population of diarrhea patients to provide scientific bases for prevention. Methods Both FTA and commercial DNA stool Mini Kit were employed for DNA extraction for cryptosporidium in five positive and three negative stools which had been confirmed by etiological examination. The methods of direct smear,iodinc staining and fluorescent PCR were used to detect cryptosporidium infection situation from stool of 63 diarrhea patients. Results The method of DNA Extraction using FTA card is superior to commercial DNA extraction kit,and 63 cases of diarrhea patients using direct smear,iodinc staining and fluorescent PCR were all negative results. Conclusion The FTA / PCR is a simple,sensitive and valid technique for detecting cryptosporidium in human stool samples,combined with a variety of detection methods to improve the detection of infection rate.