广西褐家鼠感染路氏锥虫分子生物学鉴定及系统发育分析

目的对广西部分地区褐家鼠感染的路氏锥虫进行分子生物学鉴定,分析不同地理种群间的遗传分化。方法从广西靖西、百色、南宁、天峨4地感染路氏锥虫的褐家鼠脾脏或血凝块中提取DNA,设计ITS1保守引物进行PCR扩增,将PCR产物直接或克隆测序。用DNAstar7.1进行同源性比对,用Mega4.0进行差异分析与聚类、构建系统进化树。结果广西各地血涂片阳性鼠标本均扩增出特异性DNA条带,4地样品测序结果与GenBank上登录的路氏锥虫（T.lewisi）序列的同源性均高达96%以上。区内各地理种群之间遗传距离均在0.021以下,与其他3个近缘种T.otospermophili、T.grosi、T.kuseli间遗传距离为0.15～0.206。结论广西4地鼠感染的锥虫为路氏锥虫。ITS1可以作为路氏锥虫的种属分类和分子诊断的标记。

Molacular biological identification and phylogenetic analysis of Trypanosoma lewis infection in Rattus norvegicus from several areas of Guangxi

Aim To indentify Trypanosoma.lewisi infections in Rattus norvegicus from Guangxi with molacular biological technique and differentiate rDNA-ITS1 sequences among the geographical populations of the trypanosome.Methods Genomic DNA was extracted from blood colts or spleens of R.norvegicus infected with trypanosomes from Nanning,Baise,Jingxi,Tianee in Guangxi.PCR assays with primers flanking trypanosome rDNA internal transcribed spacer 1（ITS1） were performed and the amplicons were sequenced directly or after cloning to plasmids.The rDNA-ITS1 sequences were analzed with DNAstar7.1 and the phylogenetic tree was constructed with MEGA4.0.Results All the four geographical isolates were positive with specific amplicons.The rDNA-ITS1 sequences of T.lewisi from Guangxi showed high homology（over 96%） with the sequence of T.lewisi retrieved from GenBank.Based on the ITS1 sequence the genetic distance among the geographical populations of T.lewisi was very low（less than 0.021）,But when comparing with its closely related species T.otospermophili,T.grosi and T.kuseli,the genetic distance were significantly higher（0.15~0.206）.Conclusion The trypanosome in rats from Guangxi is T.lewisi and shows less difference among geographical populations.ITS1 is one of good markers for molecular diagnosis and phylogenesis on Trypanosoma.