SYBR Green荧光定量粪检PCR法检测日本血吸虫感染的敏感性研究  

Sensitivity of SYBR Green real-time copro-PCR on detection of Schistosoma japonicum infection

作　　者：秦圆方[1] 刘云[1] 杜学利[1] 孟睿[1] 储凯[1] 季旻珺[1] Jonathan D Kurtis 吴海玮[1] 

机构地区：[1]南京医科大学病原生物学系,江苏省现代病原生物学重点实验室,江苏南京210029 [2]国际健康研究中心,美国罗德岛医院,布朗大学

出　　处：《中国病原生物学杂志》2009年第6期432-435,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30671836)

摘　　要：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 real-time Copro-PCR method to detect Schistosoma japonicum infection and to evaluate the sensitivity of this method. Methods The artificial positive fecal samples were prepared by mixing counted schistosomal eggs with fecal samples of healthy buffalos. DNA templates for SYBR Green real-time PCR reaction were extracted from the artificial positive fecal samples using QIAamp DNA Stool Kit with modified protocols. The relationship between the logarithmic transformated egg counts of artificial positive fecal samples and Ct values was studied. For quality control, quantitative PCR reactions using the DNA templates extracted from eggs only and the normal saline （without fecal samples） were also performed throughout the procedure. Results This real-time PCR method is sensitive to detecting 1 schistosomal egg in 200 mg fecal samples. A linear regression was established between Ct values and eggs per gram （EPG） of feces. Conclusion The SYBR Green real-time PCR method is a highly sensitive copro-method to detect S.japonicurn infection. A linear relationship is defined between the logarithmic transformated EPG and Ct values.

关 键 词：荧光定量PCR 粪样 血吸虫 日本 敏感性 

分 类 号：R383.24[医药卫生—医学寄生虫学]