巢式PCR检测日本血吸虫感染的研究

Detection of Schistosoma japonicum Infection by Nested-PCR

本研究通过设计2对巢式引物扩增日本血吸虫高拷贝的Sjal基因片段，建立检测日本血吸虫感染的巢式PCR技术，并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测。建立的巢式PCR方法特异性扩增日本血吸虫420bp的Sial片段，和曼氏血吸虫没有交叉，基因组DNA作为模板时最低检测量为0．1圾。小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA，建立的方法能同时检测血清和全血标本。钉螺实验感染4h后能检测到13本血吸虫DNA，现场采集钉螺的检测结果显示比传统的镜检方法敏感性高。建立的巢式PCR检测日本血吸虫感染具有较高的敏感性和特异性，为疾病诊断和媒介调查提供了新的分子生物学检测技术。

Two pairs of nested-primers were designed targeting the Schistosoma japonicum highly repeated Sial gene sequence to develop a nested-PCR for detection of Schistosoma japonicum. A 420 bp DNA fragment was specifically amplified, and the detection limit was 0.1 fg when genome DNA of S. japonicum was used as PCR template. No specific PCR product was detected with DNA from S. mansoni. Samples of experimentally infected mice and Oncomelania snails were analyzed by nested-PCR. The results showed the Sial gene of S. japonicum from serum or blood samples of infected mice was successful detected and specific DNA can be detected in mice serum samples 2 weeks after infection. For snail samples, specific DNA can be detected 4 h after experimental infection, and was more sensitive than microscopical method. The nested-PCR for detection of S. japonicum was established successfully with high sensitivity and specificity, and may constitute an alternative to the available diagnostic techniques for diagnosing S. japonicum infection.