实时荧光PCR检测蓝氏贾第鞭毛虫和隐孢子虫方法的建立

Detection of Giardia lamblia and Cryptosporidium by the real-time PCR method

目的建立贾第鞭毛虫和隐孢子虫的基因检测方法。方法根据GenBank中贾第鞭毛虫和隐孢子虫相对保守序列,设计引物及其相应的Taqman探针。通过对引物和探针浓度、Taq酶以及反应条件等优化筛选后,建立检测贾第鞭毛虫和隐孢子虫的荧光PCR方法。结果贾第鞭毛虫和隐孢子虫的检测结果为阳性,对其它DNA样本如日本血吸虫、刚地弓形虫、溶组织内阿米巴、旋毛虫和阴道毛滴虫的检测均为阴性。本法的敏感性高,可检测到10～102copies/μl的DNA浓度。结论建立的基因检测方法,对贾第鞭毛虫和隐孢子虫的检测具有高度的特异性和敏感性,可用于饮用水和临床样本等的快速检测。

Objective The present study aims to establish a real-time PCR method for the detection of Giardia lamblia and Cryptosporidium. Methods Specific primers and the corresponding Taqman probes were designed according to the conserved sequences recorded in GenBank. Results Using the designed primers and probes , positive results were obtained from the samples of Giardia lamblia and Cryptosporidium . Non-specific detection of other DNA samples , such as Schistosoma japonicum, Toxoplasma gondii, Entamoeba histolytica, Trichinella spiralis and Trichomonas vaginalis was not observed. The sensitivity of this method is 10～102 copies / μl. Conclusion The established method is specific and sensitive for the detection of Giardia lamblia and Cryptosporidium. This method could be used in the rapid detection of drinking water and clinical samples.