馆藏泽蛙标本壶菌病原实时PCR检测与系统发育分析

摘要

In order to research and prove the chytridiomycosis of amphibians in our country in the history, review the origin of the Batrachochytrium dendrobatidis from the aspects of time and systematic evolution, 39 Rana limnocharises which were collected from Guangdong province and held in museum in 1982 were screened by Taqman-MGB fluorescence probe quantitative polymerase chain reaction to detect the pathogens; and the products of QPCR were cloned and sequenced to identify the origin of the pathogens by sequence alignment and phylogenetic analysis. Finally we got the standard curve: Y = - 3.1 X + 32.65 and the related coefficient: R2 = 0.999 8. There were 12 positive samples were gotten to report the detection rate as 30.8 %. Meanwhile the phylogenetic analysis indicated that a certain extent differentia- tion of the Batrachochytrium dendrobatidis in our country existed. One type of the fungi had altitudinal genetic relationship with the strains from the North America, South America and Europe. Another one was obviously different from the strains in the other areas of the world with special characteristics. The research boosted the earliest record of the Batrachochytrium dendrobatidis in China to 1980s.%为研究、验证我国两栖类壶菌病的历史疫情,从时间和系统进化角度追溯壶菌的来源,该研究选取北京自然博物馆馆藏1982年采集于广东的泽蛙标本39只,利用Taqman-MGB荧光探针定量PCR技术进行壶菌检测,并对定量PCR产物克隆、测序,通过序列比对和系统发育分析判定其来源。最终得到定量PCR标准曲线：Y=-3.1X＋32.65;相关系数R2=0.999 8;检测结果为阳性样本12只,检出率30.8%;同时系统发育分析表明,我国的壶菌存在一定程度的分化,一类与北美洲、南美洲、欧洲菌株呈现高度的亲缘关系;另一类则表现出与世界其他地区分布的壶菌有明显的不同,显示为独特类型。该研究把我国壶菌感染的最早记录推进到了20世纪80年代初期。