无绿藻PCR ITS1-ITS2基因分型法的建立及应用

The establishment and application of PCR ITS1-ITS2 genotyping method in the identification of Prototheca strains

目的建立无绿藻(Prototheca)PCR ITS1-ITS2基因鉴定分型方法,并通过系统发育树以研究各型别无绿藻菌株间的亲缘关系。方法抽提5株无绿藻临床分离菌株的DNA,通过特异性引物扩增ITS1及ITS2区后,根据该区域核苷酸片段的差异进行基因分型,借助MEGA6.0软件建立系统发育树,并通过SSU rDNA测序加以验证。结果 5株菌株中,1株鉴定为P.zopfii hydrocarbonea,2株鉴定为P.wickerhamii。虽然由于数据库中缺乏P.zopfii portoricensis菌株ITS序列导致1、2号菌株不能被鉴定,但ITS区系统发育树中这两株菌仍被归为P.zopfii中独立的一簇,与SSU rDNA系统发育树相近,证明此分型方法无误。结论 PCR ITS1-ITS2基因分型法可作为一种有效基因分型方法用于鉴定和监测无绿藻感染。

Objective To establish the Prototheca PCR ITS1-ITS2 genotyping method,and to generate the phylogenetic tree to study the relationship between different types of Prototheca strains.Methods The extracted DNAs of 5 clinical Prototheca isolates were used to amplify ITS1 and ITS2 region through specific primers.Then,the phylogenetic tree was established with MEGA 6.0 software and verified by SSU rDNA sequencing.Results Among the 5 strains,1 strain was identified as P.zopfii hydrocarbonea,2 strains were P.wickerhamii.Although the rest two strains couldn't be identified due to the lack of ITS sequences of P.zopfii portoricensis,they were still classified as P.zopfii and in an independent cluster which was different from P.zopfii hydrocarbonea.This result was verified by SSU rDNA sequencing.Conclusions The method of PCR ITS1-ITS2 genotyping can be used as an effective genotyping method for identification and surveillance of Prototheca infection.