聚合酶链反应检测粪便中微小隐孢子虫

目的：利用聚合酶链反应（ＰＣＲ）原理建立一种敏感且特异的方法检测人和动物粪便标本中的微小隐孢子虫（Ｃｒｙｐｔｏｓｐｏｒｉｄｉｕｍｐａｒｖｕｍ，Ｃ．ｐ．）。方法：从含有Ｃ．ｐ．卵囊的人和豚鼠粪便标本中直接提取ＤＮＡ用作ＰＣＲ的模板。用一对人工合成的寡核苷酸序列作为ＰＣＲ引物，扩增长为４５２ｂｐ的Ｃ．ｐ．目的ＤＮＡ片段。扩增产物用琼脂糖凝胶电泳、溴化乙锭染色检测。结果：从感染Ｃ．ｐ．的人和豚鼠粪便标本ＤＮＡ抽提物中均扩增出目的片段，而从其他几种寄生虫、肠道微生物或宿主ＤＮＡ中均不能扩增出目的片段。本方法的敏感性比目前常用的检测方法高约１００倍。结论：ＰＣＲ技术检测人和动物粪便中的Ｃ．ｐ．，具有敏感性高且特异性强的特点，有希望成为隐孢子虫病诊断和流行病学调查的有力手段。

DETECTION OF CRYPTOSPORIDIUM PARVUM IN FECAL SAMPLES BY POLYMERASE CHAIN REACTION

AIM: To develop a sensitive and specific procedure based on the polymerase chain reaction (PCR) for detection and identification of Cryptosporidium parvum in fecal samples from infected humans and animals. METHOD: DNA extracted directly from fecal samples with C. parvum oocysts from humans and guinea pigs was served as the source of template for the PCR. A pair of oligonucleotide primers was synthesized and used to prime the amplification of a 452 bp target fragment specific for C. parvum. The PCR products were analyzed by electrophoresis in agarose gels stained with ethidium bromide. RESULTS: The target fragment was present in DNA extracts of fecal samples from humans and guinea pigs infected with C. parvum, but not in DNA from any other parasite, Candida albicans, E. coli, normal humans or guinea pigs tested. The level of detection by this method is shown to be about 100 times more sensitive than the currently used laboratory methods for diagnosis of cryptosporidiosis. CONCLUSION: The PCR assay developed is a relatively simple, highly sensitive and specific method for detection of C. parvum and could serve as a valuable tool for clinical diagnosis and epidemiological survey.