DEVELOPMENT OF AN EFFICIENT PCR METHOD FOR TOXIN TYPING OF ACTINOBACILLUS-PLEUROPNEUMONIAE STRAINS
DEVELOPMENT OF AN EFFICIENT PCR METHOD FOR TOXIN TYPING OF ACTINOBACILLUS-PLEUROPNEUMONIAE STRAINS
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DOI：
10.1016/s0890-8508(95)90158-2
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发表时间：
1995-08-01
影响因子：
3.3
通讯作者：
NICOLET, J
中科院分区：
文献类型：
--
作者：
FREY, J;BECK, M;NICOLET, J
建立了一种仅用两个PCR反应就能测定胸膜肺炎放线杆菌中三种毒素Apxl、April和ApxIII的激活基因、结构基因和分泌基因的方法。设计寡核苷酸引物以在单一PCR反应中一起扩增激活基因和结构基因apxlCA、apxllCA和apxlllCA的显著部分，得到长度不同的扩增产物，以便通过琼脂糖凝胶电泳清楚地分离。在引物的设计中考虑了在不同血清型中发现的apxlA和apxllA基因的变异，以得到apxlA和apxllA基因的两种变体的均匀扩增产物。分泌基因apxlBD和apxlllBD也在含有两对寡核苷酸引物的单一PCR反应中检测，所述引物产生两个不同大小的片段以区分apxlBD和apxlllBD基因。A.分析了胸膜肺炎血清型1-12和104个代表从世界各地不同实验室获得的所有血清型的田间菌株的APR基因含量。这两个PCR反应给出了毒素基因型，其是A.胸膜肺炎，并允许快速区分五种毒素类型组，组1包括血清型1、5a、5 b、9和11，组2包括血清型2、4、6、8，组3包括血清型3，组4包括血清型7和12，组5包括血清型10。该方法增强并促进了A.本发明提供了一种用于诊断和流行病学的胸膜肺炎菌株，并且允许检测具有非典型毒素模式的血清型。(C)1995年学术出版社
A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins Apxl, April and Apxlll in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxlCA, apxllCA and apxlllCA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxlA and apxlllA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxlA and the apxlllA genes. The secretion genes apxlBD and apxlllBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxlBD and apxlllBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apr genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns. (C) 1995 Academic Press Limited