应用FTA/PCR检测粪便中隐孢子虫的实验研究

目的 建立用FTA/PCR检测粪便中隐孢子虫的技术,并与常用的商业化的粪便DNA抽提试剂盒进行比较.方法 选择经病原学检测已经确诊的隐孢子虫阳性和阴性粪便,分别采用FTA卡和商业化DNA抽提试剂盒抽提粪便样本中DNA,进行巢式PCR扩增,对PCR阳性产物进行测序并利用BLAST在NCBI上与GenBank数据库进行比对,做同源性分析,确定虫种.结果 应用商业化DNA抽提试剂盒抽提DNA,无论是否反复冻融破壁,2个阳性对照样本扩增出目的 片段,另3个阳性样本未扩增出特异性条带.样本纯化后应用FTA卡抽提DNA进行PCR检测,所有阳性粪样均扩增出830 bp左右的目的 片段,通过测序和比对,其中2个为贝氏隐孢子虫,1个为火鸡隐孢子虫.结论 应用FTA进行粪便样本中隐孢子虫DNA的抽提方法具有简单有效、快速灵敏、准确可靠等特点,可以应用于粪便中隐孢子虫的检测.

Detection of Cryptosporidium spp.in human stool samples with FTA-PCR

Objective To develop a FTA-PCR technique for detection of Cryptosporidium spp. In human stool samples, and compare it with QIAamp DNA Stool Mini Kit-PCR. Methods Both FTA and QIAamp DNA Stool Mini Kit were employed for DNA extraction for Cryptosporidium spp.in positive and negative stools which had been confirmed by etiological examination. The SSU rRNA fragment of Cryptosporidium spp. Was amplified by nested PCR from DNA extracts of human stool samples. After purification, the gene fragments were sequenced and the sequences were determined and analyzed by BLAST. Results The amplified fragments of Cryptosporidium spp. Using FTA-PCR were about 830 bp in length, through identified in the GenBank, two of them were C. Baileyi and one of them was C. Meleagridis. Among stools that were extracted DNA by QIAamp DNA Stool Mini Kit, only two samples were amplified with positive gene fragment of Cryptosporidium spp. Conclusion The FTA-PCR is a simple, sensitive and valid technique for detecting Cryptosporidium spp. In human stool samples.