Real-time PCR法用于日本血吸虫感染宿主血清DNA的定量检测及其感染度的评价  

Quantificational detection of Schistosoma japonicum DNA in serum of the host and assessment of infectiosity by real-time PCR

作　　者：官威[1] 许静[1] 孙缓[1] 梁松[1] 董兰兰[1] 夏超明[1] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院病原生物学系,苏州215123

出　　处：《中国人兽共患病学报》2014年第3期263-267,277,共6页Chinese Journal of Zoonoses

基　　金：国家973计划项目子课题(No.2007CB513102)~~

摘　　要：目的以日本血吸虫高度重复序列-逆转录转座子SjR2为靶序列,建立TaqMan实时定量PCR法检测宿主血清中的日本血吸虫DNA用于评价感染度。方法以real-time PCR法检测不同感染度的家兔模型血清DNA。结果该法具有高度的敏感性和特异性,可以检测到44.7拷贝的重组质粒DNA。在感染家兔后的3d到7周血清中均能检测到血吸虫DNA,不同感染度早期检测的时间节点及其检测阈值分别为,家兔感染30条尾蚴(EPG=14)需在感染后2周才能检测到目的DNA,其含量为119.03拷贝,感染50条尾蚴(EPG=24)、感染100条尾蚴(EPG=48)则在感染后1周可检测到目的DNA,其含量分别为54.36拷贝和72.24拷贝,在家兔感染200条尾蚴(EPG=97)、感染500条尾蚴(EPG=232)最早在感染后3d即可检测到目的 DNA,其含量分别为60.34拷贝和142.47拷贝。日本血吸虫感染宿主血清DNA水平与感染度呈正相关,即血清DNA浓度随感染度的增大而上升。结论本研究所建立的real-time PCR法可定量检测血清DNA动态变化并对日本血吸虫病诊断及感染度的评价具有潜在的应用价值,为日本血吸虫病诊断与疗效考核提供了新方法。In this study, a TaqMan real-time PCR assay which targeted the highly repeated sequence of Schistosoma ja- ponicum （S. japonicum） retrotransposon 2 was established to detect Schistosoma japonicum DNA in serum samples of host and to evaluate infectiosity. The method had high sensitivity and specificity, being able to detect 44.7 copies standard plasmid clones of DNA. The earliest time of detection of S. japonicum DNA was at the 3^rd day post-infection, and the time points of early detection and detection threshold with different infectiosity were as follows： the target DNA in sera of rabbits infected with 30 cercariae （EPG= 14） could not be detected until the 2nd week post-infection and the DNA level was 119.03 copies; the target DNA in sera of rabbits infected with 50 cercariae （EPG=24） and 100 cercariae （EPG=48） could be detected at the 1s, week post-infection and the DNA level were 54.36 copies and 72.24 copies, respectively; the target DNA in sera of the rabbits infected with 200 cercariae （EPG=97） and 500 cercariae （EPG=232） could be detected at the 3rd day post-infection and the DNA level were 60.34 copies and 142.47 copies, respectively. Results showed that the serum DNA level of host infected with S. japonicum was positive correlation with infectiosity. The established TaqMan real-time PCR assay, as a sensitive, specific and convenient method, could quantificationally detect dynamic changes of serum DNA, and has potential application value in diagnosis of schistosomiasis and evaluation of infectiosity, providing a new assay for schistosomiasis diagnosis and evaluation of chemotherapy.

关 键 词：日本血吸虫 TaqMan实时定量PCR 血清DNA 感染度 检测阈值 

分 类 号：R383.2[医药卫生—医学寄生虫学]