通过16S-23S基因间转录间隔区PCR和限制性片段长度多态性分析快速鉴定和区分软腐欧文氏菌。
Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses.
作者信息
Toth I K, Avrova A O, Hyman L J
机构信息
Unit of Mycology, Bacteriology and Nematology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.
出版信息
Appl Environ Microbiol. 2001 Sep;67(9):4070-6. doi: 10.1128/AEM.67.9.4070-4076.2001.
Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the alpha-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten "atypical" E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two "atypical" E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.
目前对软腐欧文氏菌的鉴定方法既不精确又耗时。我们利用16S - 23S rRNA基因间转录间隔区（ITS）来辅助对它们进行鉴定。与当前的分子和表型技术相比，ITS - PCR及ITS - 限制性片段长度多态性分析被发现是一种简单、精确且快速的方法。使用通用PCR引物从欧文氏菌属及其他属中扩增ITS。PCR后，产生的条带模式可使软腐欧文氏菌与所有其他欧文氏菌属和非欧文氏菌属物种区分开来，并归入三个组（I至III）之一。I组包括所有胡萝卜软腐欧文氏菌黑腐亚种和甜菜维管束亚种分离株。II组包括所有胡萝卜软腐欧文氏菌胡萝卜亚种、香味亚种、辣根亚种和仙人掌欧文氏菌分离株，III组包括所有菊欧文氏菌分离株。为了进一步提高鉴别水平，用两种限制性内切酶之一对ITS - PCR产物进行消化。用CfoI消化可鉴定胡萝卜软腐欧文氏菌黑腐亚种和甜菜维管束亚种（I组）以及菊欧文氏菌（III组）分离株，而用RsaI消化可鉴定胡萝卜软腐欧文氏菌辣根亚种、胡萝卜亚种、香味亚种/胡萝卜亚种和仙人掌欧文氏菌分离株（II组）。在后一种情况下，有必要使用α - 甲基葡萄糖苷试验来区分胡萝卜软腐欧文氏菌香味亚种和胡萝卜亚种。从澳大利亚分离出的60株疑似软腐欧文氏菌被鉴定为胡萝卜软腐欧文氏菌黑腐亚种、菊欧文氏菌、胡萝卜软腐欧文氏菌胡萝卜亚种和非软腐物种。10株“非典型”胡萝卜软腐欧文氏菌黑腐亚种分离株被鉴定为胡萝卜软腐欧文氏菌黑腐亚种、胡萝卜亚种、甜菜维管束亚种和非软腐物种，2株“非典型”胡萝卜软腐欧文氏菌胡萝卜亚种分离株被鉴定为胡萝卜软腐欧文氏菌胡萝卜亚种和黑腐亚种。
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