聚合酶链反应检测日本血吸虫DNA的实验研究

Detection of Schistosoma japonicum DNA by polymerase chain reaction

目的探索核酸检测在日本血吸虫病诊断中的应用。方法根据日本血吸虫基因设计特异性引物,采用PCR方法对日本血吸虫的成虫、虫卵、日本血吸虫感染家兔的肝组织、粪便、外周血清标本中的DNA进行扩增,并将虫卵和粪便的模板DNA倍比稀释后进行扩增,以测定PCR扩增法的敏感性。结果从日本血吸虫的成虫、虫卵、日本血吸虫感染家兔的肝组织、粪便和外周血清中均扩增到分子量为230bp的阳性条带,扩增产物经测序并在基因库中匹配,证实为日本血吸虫基因中的一个片段,其同源性为98%。其PCR扩增敏感性检测结果表明,0.021个虫卵DNA模板即可扩增出该特异性阳性条带。结论聚合酶链反应检测日本血吸虫DNA有较高的敏感性,尤其是能从日本血吸虫感染宿主血清中检测出特异性DNA,具有潜在的诊断应用价值。

In order to develop a method to detect the presence of nucleic acids in Schistsomajaponicum, specific primers were designed based on the genome of S. japonicum and then PCR assay was used to detect DNAs from samples of eggs, adult worms, liver feces and peripheral blood of rabbits infected with S. japonicum. Meanwhile, the template DNAs of eggs and feces were diluted before amplification in order to test the sensitivity of the PCR assay. A 230 bp DNA fragment was obtained from all the samples being tested, and then the gene product was sequenced and matched in GenBank. It was confirmed that this product was a fragment of S. japonicum gene. Result in the PCR amplification sensitivity testing revealed that 0. 021 egg DNA template would be enough to amplify this specific positive band. It is evident form the above that the detection of S. japonicum DNA by PCR exhibits higher sensitivity, especially to detect the specific DNAs from serum of hosts infected with S. japonicum, thus providing potential diagnostic values.