检测水体日本血吸虫毛蚴PCR方法的建立  

Establishment of PCR assay for detection of Schistosoma japonicum miracidium in water

作　　者：王廷安 沈海英[1] 申云侠[1] 王文波[1] 王本敬[1] 梁幼生[2] 诸葛洪祥[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 [2]江苏省血吸虫病防治研究所,无锡214064

出　　处：《中国人兽共患病学报》2010年第6期562-564,共3页Chinese Journal of Zoonoses

基　　金：江苏省自然科学基金(BK2009076)资助

摘　　要：目的建立一种灵敏、特异的痕量检测日本血吸虫毛蚴的PCR方法。方法取感染6～8w日本血吸虫的小鼠肝脏,碾磨后沉淀,孵化并利用间接连续离心法富集毛蚴,采用蛋白酶K-苯酚法提取毛蚴基因组DNA。登陆GenBank查询获得日本血吸虫保守序列18S小亚基单位核糖体脱氧核酸(18SrDNA)基因,利用引物设计软件PrimerPremier5.0和Oligo6自主设计一对引物,建立检测日本血吸虫毛蚴的PCR方法。梯度稀释血吸虫毛蚴基因组DNA进行PCR方法的灵敏性试验,设置阴性对照,PCR产物通过电泳鉴定。结果 PCR扩增日本血吸虫毛蚴得到特异性DNA片段,片段长度为463bp,阴性对照组无扩增产物。PCR法可检测出日本血吸虫毛蚴DNA的最低浓度为62.5pg/μL。结论建立的检测日本血吸虫毛蚴PCR方法灵敏、特异,具有一定的预警作用。To establish a sensitive and specific polymerase chain reaction（PCR）assay for detecting Schistosoma japonicum（S.japonicum）miracidium,eggs of S.japonicum were harvested from infected mice livers 6 to 8 weeks after infection and incubated with water as well as collected consecutively by centrifugalization.The DNA of S.japonicum miracidium was extracted by proteinase K/phenol method.Based on 18S small subunit ribosomal DNA（18S SSU rDNA）gene of S.japonicum in GenBank,a PCR assay for detecting miracidium of S.japonicum was established by Primer Premier 5.0 and Oligo 6.Sensitivity of PCR assay was tested by gradient dilution of miracidium DNA.The negative control was set and the PCR products were identified by electrophoresis.PCR results were judged with naked eyes and electrophoretic analysis,respectively.The positive signal with an approximate length of 463bp was observed in miracidium of S.japonicum.By contrast,no positive signal was observed in negative control.Experiments showed that the minimum DNA concentration of S.japonicum miracidium was 62.5 pg/μL.It＇s suggested that PCR assay for detection of S.japonicum miracidium is high in sensitivity and specificity.

关 键 词：日本血吸虫 毛蚴 PCR 痕量检测 预警 

分 类 号：R383.2[医药卫生—医学寄生虫学]