巢式PCR法在日本血吸虫易感水域哨鼠监测中的应用  

The use of nested PCR to monitor sentinel mice in aquatic areas infested with Schistosomiasis japonica

作　　者：张凡[1] 侯敏[1] 余传信[2] 杨坤[2] 杨丙雅 张文月[1] 骆晓凤 陈琳[1] 季旻珺[1] 

机构地区：[1]南京医科大学病原生物学系,江苏南京210029 [2]卫生部重要寄生虫病预防与控制高技术重点实验室,江苏省血吸虫病防治研究所,江苏无锡214064

出　　处：《中国病原生物学杂志》2015年第4期325-328,共4页Journal of Pathogen Biology

基　　金：国家十一五科技重大专项(No.2008ZX10004-011);国家十二五科技重大专项(No.2012ZX10004220)

摘　　要：目的建立巢式PCR法用以辅助日本血吸虫易感水域中哨鼠的监测。方法选择日本血吸虫基因组重复序列DNA SjR2（2456～3171bp）作为外道PCR的区域,其中2719～3019bp作为内道PCR的扩增区域。以不同稀释度的日本血吸虫成虫基因组DNA为模板进行敏感性测试,并对未感染和感染10条、40条尾蚴的小鼠血清进行检测。将巢式PCR法用于2013年8个哨鼠监测点的监测,观察其应用价值。结果巢式PCR中内道引物扩增产物为301bp,血吸虫基因组DNA浓度低至10-5ng/μl时,仍扩增阳性;以10条和40条尾蚴感染4周的小鼠血清DNA为模板进行巢式PCR,结果为阳性。将巢式PCR用于2013年血吸虫易感水域小范围哨鼠的监测,第2组的20只小鼠中有2只阳性,但解剖小鼠未检出虫体。结论巢式PCR法可辅助血吸虫易感水域中哨鼠的监测,特别对感染度较低水域的监测有一定帮助。Objective To establish a method of nested PCR to monitor sentinel mice in aquatic areas infested with Schistosoma japonicurn. Methods The sequence of a highly repetitive retrotransposon of S. japonicurn, SjR2 （2 456--3 171 bp）, was amplified using outer primers. A SjR2 fragment of 2 719 bp to 3 019 bp was amplified again using inner primers. Different dilutions of S. japonicurn DNA served as a template for a sensitivity test. Sera from uninfected mice and mice infected with 10 or 40 cercariae were tested using nested PCR. This method was used to monitor sentinel mice at 8 surveillance sites in 2013. Results PCR amplification with inner primers resulted in a product that was 301 bp in length. Nested PCR detected S. japonicum DNA at concentrations as low as 10--5 ng/μl. Nested PCR also detected S. japonicurn in sera from mice infected with 10 or 40 cercariae after 4 weeks. When nested PCR was used to monitor aquatic areas infested with S. japonicum, this method detected schistosomiasis in 2 of 20 mice at 8 surveillance sites. However, no parasites were found in these 2 mice. Conclusion A method of nested PCR was established and it can be used to monitor sentinel mice in aquatic areas infested with S. japonicurn.

关 键 词：血吸虫 日本 巢式PCR 哨鼠 

分 类 号：R373.21[医药卫生—病原生物学]