饮用水中贾第鞭毛虫和隐孢子虫的数字PCR测定法

Digital PCR for quantification of Cryptosporidium and Giardia in drinking water

目的建立饮用水中贾第鞭毛虫和隐孢子虫的数字PCR定量检测方法。方法过滤水样,收集贾第鞭毛虫和隐孢子虫,提取DNA,分别选择β-Giardin和18S r RNA基因作为靶基因,设计合成特异性引物与探针,使用数字PCR技术进行检测,计算两种靶基因的浓度,进而对贾第鞭毛虫和隐孢子虫个数进行定量。结果数字PCR法的检出限为0.02~0.03个/L,加标回收率>20%,RSD为12.81%~15.35%。结论该方法具有较高的灵敏度和特异性,可应用于饮用水中极少数量贾第鞭毛虫和隐孢子虫的测定。

Objective To establish a method for quantifying Cryptosporidium and Giardia in drinking water by digital PCR.Methods The water samples were filtered to collect Giardia and Cryptosporidium.Thenβ-Giardin and 18S rRNA genes were selected as the target genes and the specific primers and probes were designed and synthesized.With the digital PCR technology,the concentration of the two targets was determined and the number of Giardia and Cryptosporidium was quantified.Results The detection limits of digital PCR method were 0.02-0.03/L,the spiked recovery rates were above 20%and the RSDs were 12.81%-15.35%.Conclusion This method has high sensitivity and specificity and is applicable to the determination of a small number of Giardia and Cryptosporidium in drinking water.