摘要
目的建立两种机会性寄生原虫——微小隐孢子虫和蓝氏贾第鞭毛虫(贾第虫)基因检测方法。方法从微小隐孢子虫和蓝氏贾第鞭毛虫感染者粪便标本内分离纯化卵囊和包囊,提取基因组DNA。根据微小隐孢子虫18S rRNA基因和贾第虫磷酸丙糖异构酶(tim)基因各设计或合成两对特异性引物,采用PCR技术分别对从卵囊和包囊提取的和6种对照。DNA样本(日本血吸虫、刚地弓形虫、溶组织内阿米巴、旋毛虫和阴道毛滴虫以及人体血细胞DNA等),以及此二种虫相互间的DNA样本进行检测,以确定该法的特异性和敏感性。方法建立后,取艾滋病患者粪便标本对之进行检测。结果从微小隐孢子虫和贾第虫的DNA样本中分别扩增出各自相应基因的500 bp和683 bp长度的片段;最少可检测出20 pg隐孢子虫和0．4 pg贾第虫DNA;几种对照DNA样本均不发生交叉反应;受检的30例艾滋病患者粪便标本中,7例显示微小隐孢子虫阳性,未检出贾第虫。结论建立的基因检测方法,对微小隐孢子虫和贾第虫的检测具有高度的特异性和敏感性,可用于临床艾滋病合并感染的早期诊断和人群流行病学筛查。
Objective To establish genetic method in detecting Cryptosporidium parvum and Giardia larnblia which often coinfected with AIDS patients. Methods Cryptosporidium oocysts and Giardia cysts were isolated and purified from fecal samples of the individuals infected with C. parvum and （5. lamblia ,respectively. Genomic DNAs were extracted. Two pairs of specific primers were designed or synthesized according to the 18S rRNA gene from C. parvum or the triose phosphate isomerase （ tim ） gene from （5. larnblia. Polymerase chain reaction（PCR） technique was used to amplify the DNA samples from the oocysts and the cysts, and those from the 6 control samples, including Schitosorna japonicum, Toxoplasma gondii, Entarnoeba histolytica, Trichinella spiralis, Trichomonas vaginalis and human blood cells. DNA samples from 30 fecal samples of AIDS patients were detected with the same method. Results One fragment of 500 bp was amplified with the primer of C. parvum, and the other one of 683 bp was amplified with the primer of G. lamblia. Twenty pg and 0.4 pg DNA of C. parvum and G. lamblia could be detected separately. The specificity of these two pairs of PCR primers was confirmed by the failure in the amplification of the control DNA samples. Out of 30 cases of AIDS patients, 7 showed C. parvum positive, while non Giardia was detected. Conclusion Genetic detection method for C. parvum and （5. lamblia detection was established which was more sensitive and specific.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2006年第10期884-888,共5页
Chinese Journal of Epidemiology
基金
北京市自然科学基金资助项目(7992002)
