饮用水中隐孢子虫qPCR检测研究

Cryptosporidium qPCR assay in drinking water

分子生物学的发展推动了分子技术在病原体检测诊断中的应用,建立成熟可靠的饮用水中隐孢子虫的分子检测方法也迫在眉睫.本研究直接提取饮用水浓缩液DNA,对隐孢子虫卵囊目的基因进行定量PCR(quantitative PCR,简称qPCR)检测,并对该体系进行了综合优化.本研究比较了不同冻融处理及不同DNA提取试剂盒对隐孢子虫卵囊DNA提取效果,结果显示,QIAamp DNA Mini kit配合冻融前处理(液氮/沸水5个循环)提取DNA含量较高,用这种方法可以提取到(4±1)个隐孢子虫卵囊DNA.本研究比较了隐孢子虫SYBR green染料法及Taqman探针法qPCR,结果显示,探针法和染料法均可以检测到10个18S rRNA基因拷贝(一个隐虫卵囊含有20个18S rRNA基因拷贝).采用该检测体系测定的去离子水和水源水中隐孢子虫的加标回收率分别达到了55%和46%;本研究提供了饮用水中隐孢子虫检测的一种可行性分子方法.

The molecular methods and technologies facilitate the pathogen diagnostic ,the methodology of Cryptosporidium molecular detection in drinking water should be set up urgently. This study established the method of qPCR（ quantitative PCR）assay for Cryptosporidium ,and the whole protocol from DNA isolation to gene quantification was tested and optimized. This study compared four DNA extraction methods of Cryptospoidium oocysts, and the method 3 with five freeze-thaw cycles using liquid nitrogen and boiling water was deemed the most desirable method. （ 4＋ 1 ） oocysts could be detected with method 3. The primer set JVAF/JVAR was found specific for Cryptosporidium and selected to quantify oocysts, both standard curves fromSYBR Green qPCR and Taqman qPCR detected 10 copies of the 18S rRNA gene, corresponding to half of oocysts. Based on the protocol, the recovery of Cryptosporidium in purified water reached 55% and in resource water reached 46%. The whole qPCR assay should be a promising approach to quantify Cryptosporidium in drinking water.