PCR技术检测棘阿米巴26S核糖体DNA

Study of utility PCR amplification of gene fragment of 26s-rDNA in early diagnosis of acanthamoeba keratitis

目的:用聚合酶链反应(PCR)方法检测棘阿米巴的26S核糖体DNA(26S rDNA,Rns),为早期诊断棘阿米巴角膜炎提供方法。方法:分离培养三株不同基因型棘阿米巴虫株,提取虫株基因组DNA,合成棘阿米巴属特异性引物(ArDNA-a),通过聚合酶链反应(PCR)扩增部分26SrDNA序列。结果:三株不同基因型棘阿米巴虫株属于两种基因型,其中Acanthamoeba sp.CJY/s2株和Acanthamoeba sp.CJY/s株属于Rns T4基因型,Acastellanii Neff株属于Neff基因型。PCR扩增后,Acanthamoeba sp.CJY/s2和Acastellanii Neff株的26S核糖体DNA基因片段扩增出分子量大小为126 bp的特定扩增带,而Acanthamoeba sp.CJY/s株的26S核糖体DNA基因片段则未得到预期的扩增带。结论:用棘阿米巴属特异性引物(ArDNA-a)可以有效扩增Acanthamoeba sp.CJY/s2和Acastellanii Neff株的26S核糖体DNA基因,可以为部分棘阿米巴性角膜炎的早期检测提供新的检测手段。

Objective： To provide a method for early diagnosis of acanthamoeba keratitis using the polymerase chain reaction （PCR） amplificat the gene 26s-rDNA fragment for different species of acanthamoeba. Methods： Isolated culture 3 groups of different genotype acanthamoeba, which whole genome were extracted, PCR amplificated parts of gene sequence of 26s-rDNA by using acanthamoeba specificity primer （ArDNA-a）. Results： In 3 different species acanthamoebas, Acanthamoeba sp. CJY/s2 and Acanthamoeba sp. CJY/s were Rns genotype T4, Acastellanii Neff was Neff. After PCR, 26s-rDNA fragment about 126 base pairs was successfully amplified by single PCR from the genotype DNA of Acanthamoeba sp.CJY/s2 and Acastellanii Neff, but no anticipate fragment was obtained from the genotype DNA of Acanthamoeba sp. CJY/s. Conclusion： When utility the acanthamoeba specificity primer （ArDNA-a）,PCR can amplified gene fragment of 26s-rDNA in Acanthamoeba sp. CJY/s2 and Acastellanii Neff, which can prove a new detect method to early diagnosis of some acanthamoeba keratitis.