实验感染C57 BL/6N小鼠粪内蓝氏贾第鞭毛虫tim基因检测

建立蓝氏贾第鞭毛虫感染动物模型 ,用PCR技术对感染动物粪内该虫tim基因进行检测 ,探讨蓝氏贾第鞭毛虫基因检测方法。用人源蓝氏贾第鞭毛虫河北株 (CD)和江苏株 (XZ)的包囊分别接种两组C5 7BL 6N小鼠 ,于接种后第 6天、1 0天和 1 4天收集粪便标本 ,采用酚 氯仿法提取总DNA。将提取液分别按 1 0 0 、 1 0 - 1 、 1 0 - 2 和 1 0 - 3稀释并纯化。根据该虫磷酸丙糖异构酶 (Triosephosphateisomerase,tim)基因合成 1对特异性引物 ,采用PCR技术分别扩增各个稀释度的样本。结果贾第虫阳性样本均被扩增出相应基因的 1条 683bp长的片段 ,纯化后的各个稀释度样本的PCR检测阳性率随稀释倍数的增加而增高。本研究结果为贾第虫感染基因检测方法的进一步研究提供了实验资料。

DETECTING TIM GENE OF GIARDIA LAMBLIA IN THE STOOL SPECIMENS FROM C57BL/6N MOUSE INFECTED EXPERIMENTALLY

To approach a genetic detecting method of Giardia lamblia infection,a C57BL/6N mouse model was established.The tim gene of G.lamblia in the stool specimens of the inoculated animal was detected with PCR.Two groups of mice were orally inoculated with cysts of two human origin isolates (CD and XZ) separately.After inoculation,stool specimens of the infected mice were collected on day 6,10 and 14.Cysts were isolated and purified using sucrose-gradient-centrifugal method.Genomic DNAs were then extracted with phenyl-chloroform method.A pair of specific primer was synthesized according to the triose phosphate isomerase(tim) gene sequence from G.lamblia.Samples of the stool extractions from inoculated mice were diluted into a series of 10 0,10 -1 ,10 -2 and 10 -3 and purified.PCR positive results were determined as one fragment of 683bp was obtained after PCR amplification.PCR positive rate increased along with the increased dilution of the plate DNA.The present results supplied useful information for the further investigation of the genetic detection of Giardia lamblia infection.