摘要
目的建立灵敏、特异的检测日本血吸虫感染性钉螺的PCR方法。方法根据日本血吸虫18S小亚基单位核糖体核酸(18S-rRNA)基因设计1对引物,建立检测日本血吸虫感染性钉螺的PCR方法。测定PCR产物的DNA序列,稀释血吸虫毛蚴DNA进行PCR方法的灵敏性试验,扩增单尾尾蚴感染的钉螺DNA进行交叉反应试验,并根据不同稀释度的感染性钉螺DNA的扩增结果来验证PCR的群体检测效果。结果PCR扩增日本血吸虫感染性钉螺,得到了与靶DNA片段位置相同的产物,测序片段长度为469bp,与靶DNA相同且序列一致,并将序列登录GenBank(注册号:DQ442999)。扩增阴性钉螺无产物出现。灵敏性试验提示,PCR可检出日本血吸虫毛蚴DNA的最低浓度为40pg/μl;交叉反应试验显示,PCR方法不能扩增出单尾尾蚴感染钉螺的DNA;群体检测试验表明,PCR可检出感染性钉螺提取的DNA最高稀释度为1∶640。结论初步建立的检测日本血吸虫感染性钉螺的PCR方法灵敏、特异且具有良好的群体检测效果。
Objective To establish a sensitive and specific PCR assay for detecting Schistosoma japonicum- infected Oncomelania hupensis. Methods Based on 18S-rRNA gene of S. japonicum, a PCR assay for detecting Oncomelania snails infected with S. japonicum was established. The PCR product was sequenced, and the sensitivity, crossreaction and mass detection experiments of PCR assay were performed. Results The location of PCR product for detecting Oncomelania snails infected with S. japonicum was similar to the target DNA, with a length of 469 bp and the same sequence as the target DNA. It was registered in GenBank （Accession No. DQ442999）. There was no PCR product for detecting uninfected snail. Experiments showed that the minimum DNA concentration of S. japoncium miracidium to be detected was 40 pg/μl. DNA from snail infected with single-tail cercaria could not be detected. The maximum dilution concentration of infected snail DNA pooled with uninfected snail DNA that could be detected was 1：640. Conclusion The PCR assay for detecting S. japonicum-infected Oncomelania snails shows high sensitivity, specificity and effect of mass detection.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2006年第3期204-207,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
浙江省医药卫生科学研究基金(No.2004B001)~~
