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目的建立快速、敏感、特异的检测日本血吸虫感染的SYBR Green荧光定量PCR法,准确评估其敏感性。方法将计数的日本血吸虫虫卵掺入健康水牛粪样中,制备人工阳性粪样。采用改良QIAamp DNA Stool Kit粪样DNA提取方法,提取人工阳性粪样DNA,进行SYBR Green荧光定量PCR,建立Ct值与粪样中克粪虫卵数(EPG)的关系;提取单独(不与牛阴性粪样混合)日本血吸虫虫卵DNA与虫卵生理盐水冲洗液DNA,行定量PCR检测,以对本方法进行严格质量控制。结果每200mg粪样仅含1个虫卵时,荧光定量PCR仍呈阳性,人工阳性粪样EPG的对数与PCR的Ct值存在线性关系。结论SYBR Green荧光定量粪检PCR法检测日本血吸虫虫卵DNA敏感性高,EPG对数与PCR的Ct值存在线性关系。
Objective To establish a rapid, sensitive and specific SYBR Green fluorescence quantitative PCR method for the detection of Schistosoma japonicum infection and evaluate its sensitivity. Methods The counted Schistosoma japonicum eggs were mixed into healthy buffalo dung samples to prepare artificial positive fecal samples. A modified QIAamp DNA Stool Kit was used to extract artificial positive stool DNA, and SYBR Green fluorescence quantitative PCR was used to establish the relationship between Ct value and egg count (EPG) in fecal samples. Negative fecal samples were mixed) Schistosoma japonicum egg DNA and egg normal saline wash DNA, quantitative PCR detection, in order to strictly control the quality of the method. Results Fluorescence quantitative PCR was still positive when there was only one egg per 200 mg fecal sample. There was a linear relationship between the logarithm of artificial positive fecal sample EPG and the Ct value of PCR. Conclusion SYBR Green fluorescence quantitative fecal PCR method for the detection of Schistosoma japonicum eggs DNA sensitivity, EPG logarithm and PCR Ct values exist a linear relationship.