微小隐孢子虫的基因检测实验研究

目的　探讨聚合酶链反应 (PCR)检测微小隐孢子虫 (Cryptosporidium parvum)感染的特异性和敏感性。　方法用 Sheather's蔗糖梯度离心法从微小隐孢子虫感染者粪便标本中分离纯化卵囊。用酚 -氯仿法抽提卵囊总 DNA。针对本虫 18S r RNA基因片段设计 1对引物 ,对模板 DNA进行 PCR扩增 ,同时以蓝氏贾第鞭毛虫 (Giardia lamblia)、溶组织内阿米巴 (Entamoeba histolytica)、阴道毛滴虫 (Trichomonas vaginalis)、刚地弓形虫 (Toxoplasma gondii)、日本血吸虫(Schistosoma japonicum)和旋毛虫 (Trichinella spiralis) ,以及人体血细胞等 DNA样本为对照。用琼脂糖电泳和溴乙腚染色对 PCR产物进行检测。　结果　仅微小隐孢子虫 DNA被扩增出一 5 0 0 bp的 DNA片段。其它对照 DNA样本均未出现扩增反应。　结论　PCR对隐孢子虫检测的特异性可高达 10 0 % ,敏感性也很强 ,可检测到 2 0 pg微小隐孢子虫卵囊的DNA。表明本实验建立的检测微小隐孢子虫的 PCR方法有效

EXPERIMENTAL STUDY ON GENE DETECTION OF CRYPTOSPORIDIUM PARVUM

Objective To approach the specificity and the susceptibility of polymerase chain reaction (PCR) for detecting the infection of Cryptosporidium parvum . Methods Sheather's sucrose gradient centrifugation method was used to isolate and purify the oocysts from the stool samples of an individual infected by C. parvum . Genomic DNA of the C. parvum oocysts was extracted with phenol/chloroform. Oligonucleotide primers were designed based on the DNA fragment of the 18S rRNA gene from C. parvum . Plate DNA was amplified with primers in PCR. The control DNA samples of Giardia lamblia, Toxoplasma gondii, Entamoeba histolytica, Trichomonas vaginalis, Trichinella spirallis, Shistosoma japonicum as well as that of human blood cells were amplified simultaneously. PCR products were detected with agar electrophoresis and ethidium bromide. Results Among the DNA samples only a fragment of 500 bp of C. parvum was amplified specifically, while no amplifications were found with the control DNA samples. Conclusion The test developed in the present experiment was more effective in the detection of C. parvum .