实验感染昆明鼠粪便微小隐孢子虫18SrRNA基因检测  

DETECTION OF CRYPTOSPORIDIUM PARVUM IN STOOL SPECIMENS OF EXPERIMENTALLY INFECTED KM MICE BY 18SrRNA GENE AMPLIFICATION

作　　者：卢思奇[1] 罗晓冰[2] 陈小宁[2] 王凤云[1] 

机构地区：[1]首都医科大学细胞生物学系寄生虫学教研室,北京100054 [2]承德医学院寄生虫学教研室,河北承德067000

出　　处：《中国寄生虫病防治杂志》2003年第4期196-199,共4页Chinese Journal of Parasitic Disease Control

基　　金：北京市自然科学基金(No.7992002)

摘　　要：目的 探讨隐孢子虫感染的基因检测方法。方法 用分离自人和牛的微小隐孢子虫各1株分别感染2组昆明小鼠,于接种后第6d、10d和14d分别收集每只动物粪便,用显微镜检查,再用Sheather's蔗糖梯度密度离心法纯化卵囊,提取总DNA,对每份DNA样本做10~0～10^(-3)稀释,然后用PCR对微小隐孢子虫18SrRNA基因进行特异性扩增。结果 接受具有活力的1×10~4个卵囊/只的36只小鼠,均获得感染;阳性样本被扩增出1条500hp的片段。隐孢子虫感染小鼠粪便样本各稀释度的PCR检测实验结果表明,PCR检测阳性率随模板DNA样本稀释倍数的增加而增高;小鼠感染后第6d、10d和14d的10~0和10^(-1)两个稀释度的PCR检测阳性率低于镜检结果,而10^(-2)和10^(-3)两个稀释度均高于镜检结果。结论 受染昆明鼠粪便内的微小隐孢子虫可通过扩增其18SrRNA基因检出。Objective To investigate genetic detection method of the infection with Cryptosporidium parvum. Methods Kun-ming mouse was used as an infected animal modei of C. parvum. Two groups of the animals were inoculated with oocysts of two dif-ferent C. parvum isolates from human (XZ) and cattle (CC) by oral gavage respectively. Stool specimens of the inoculated mice were collected on day 6, 10 and 14 post inoculations (P. I). Oocysts were purified from the stool specimens with the Sheather's su-crose-gradient-centrifugal method. Genomic DNA were extracted and diluted into 10° , 10-1 , 10-2 and 10-3 with deionized water. 18SrRNA gene was amplified specifically by PCR technique. Positive rates of PCR were compared with those examined microscopi-cally. Results All of thirty-six mice receiving 1×104 oocvsts each were infected. PCR positives were determined by the amplifica-tion of a fragment of 500 bp. The positive rates of PCR detection increased along with the increase of the dilutions of the plate DNA samples. On day 6,10 and 14 after inoculations, the positive rates of PCR detection of 10°and 10-1 dilutions were lower than the re-suits obtained with microscopic examinations, while those of 10-2 and 10-3 dilutions were higher than that examined microscopically. Conclusion The results obtained in the present study provide experimental basis for the genetic detection of C. parvum infection in the clinic.

关 键 词：昆明小鼠 粪便 微小隐孢子虫 18SrRNA基因 机会性致病性原虫 

分 类 号：R382.3[医药卫生—医学寄生虫学]