聚合酶链反应结合非放射性标记探针检测粪便中微小隐孢子虫

DETECTION OF CRYPTOSPORIDIUM PARVUM IN FECAL SAMPLES USING POLYMERASE CHAIN REACTION COMBINED WITH NON RADIOACTIVE LABELED DNA PROBE HYBRIDIZATION *

目的：利用非放射性标记探针技术检测经聚合酶链反应扩增后的人粪便中微小隐孢子虫ＤＮＡ，观察该方法的特异性和敏感性。方法：用裂解法从粪便标本直接制备微小隐孢子虫模板ＤＮＡ，用一对人工合成的寡核苷酸作ＰＣＲ引物，扩增长度为４５２ｂｐ的微小隐孢子虫目的ＤＮＡ片段。将扩增产物直接点在或经Ｓｏｕｔｈｅｒｎ印迹法转移到硝酸纤维素膜上，再和生物素标记的寡核苷酸探针杂交，经底物（ＢＣＩＰ）呈色观察。结果：阳性杂交信号只见于微小隐孢子虫ＤＮＡ扩增产物，而约氏疟原虫、杜氏利什曼原虫、贾第虫、白色念珠菌、大肠杆菌和人白细胞ＤＮＡ均无杂交信号。ＰＣＲ结合斑点杂交或Ｓｏｕｔｈｅｒｎ印迹法检测隐孢子虫ＤＮＡ的敏感性均为０．１ｐｇ。结论：非放射性探针检测人粪便隐孢子虫ＤＮＡ的ＰＣＲ扩增产物，具有敏感性高、特异性强、操作较简便及无放射性污染等优点。

AIM: To develop a non radioactive labeled probe hybridization technique for the detection of the PCR products of Cryptosporidium parvum DNA in fecal samples and evaluate its specificity and sensitivity. METHODS: DNA of C.parvum was prepared directly from C.parvum infected patients fecal samples by the direct lysis method, and was used as the template for 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 directly spotted or transferred on to NC membrane. Then, the PCR products were hybridized with the biotin labeled oligonucleotide probe and coloured by a substrate BCIP (5 bromo 4 chloro 3 indolyl phosphate). RESULTS: Positive hybridized signals were only showed in the PCR products of DNA extracted from the fecal samples of C.parvum infected patients, but not in the other DNA of P.yoelii, L.donovani, G.lamblia, C.albicans, E.coli and human leucocytes. The lowest detectable amount of both dot or Southern blotting hybridization was 0 1 pg of C.parvum DNA. CONCLUSION: A combination of PCR and non radioactive biotin labeled probe hybridization assay is highly sensitive, specific, and relatively simple and safe for the detection of the PCR products of C.parvum in fecal samples.