Use of PCR for rapid identification of Acidovorax avenae and A. avenae subsp. citrulli.
Use of PCR for rapid identification of Acidovorax avenae and A. avenae subsp. citrulli.
复制标题
使用 PCR 快速鉴定燕麦酸食菌和燕麦食酸菌亚种。
DOI：
10.1007/978-94-017-0133-4_59
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
N. Schaad
中科院分区：
文献类型：
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作者：
W. Song;A. Sechler;E. Hatziloukas;H. M. Kim;N. Schaad
燕麦食酸菌（Acidovorax avena）含有对几种作物致病的细菌：A.燕麦亚种感染燕麦、玉米、水稻、小米和甘蔗;瓜类亚种感染西瓜和甜瓜;而卡特兰亚种感染兰花。因为这些细菌是密切相关的，它们的正确识别需要昂贵，耗时的致病性测试。为了开发快速鉴定所有物种和亚种的PCR方法。瓜类，我们测序的16 S-23 S rDNA的基因间隔区和设计的davenae特异性和亚种。瓜类特异性的经典引物和实时引物和探针。经典的燕麦特异性引物Oafl/Oarl扩增了所有46株来自狗尾草、燕麦、玉米、水稻、甘蔗和小米的燕麦亚种、来自兰花的卡特利亚亚种和来自西瓜的11株西瓜亚种的DNA产物。用燕麦特异性引物Oaf 1/Oar 2和探针AaP 1对40株燕麦亚种、8株瓜类亚种和8株卡特利亚亚种进行检测。经典瓜类特异性引物Aacf 2/Aacr 2和实时引物Aacf 3/Aacr 2及探针AaP 2均能扩增出11个瓜类亚种的PCR产物，但不能扩增出其它任何一个燕麦亚种的PCR产物。超过50种其他细菌测试生成的PCR产物与经典引物或实时引物和探针。经典引物Oaf 1/Oar 1、实时荧光引物Oaf 1/Oar 2和探针AaP 1可用于A.瓜类特异性引物Aacf 2/Aacr 2、实时荧光引物Aacf 3/Aacr 2和探针AaP 2可用于瓜类亚种的特异性鉴定。
The speciesAcidovorax avenaecontains bacteria pathogenic to several crops: A. a.subsp.avenaeinfects oats, corn, rice, millet, and sugarcane; subsp.citrulliinfects watermelon and melon; and subsp.cattleyaeinfects orchid. Because these bacteria are closely related their proper identification requires expensive, time-consuming pathogenicity tests. To develop PCR assays for rapid identification of all species and subsp.citrulli, we sequenced the intergenic spacer region of the 16S–23S rDNA and designedavenae-specific and subsp.citrulli-specific classical primers and real-time primers and probes. Classicalavenae-specific primers Oafl/Oarl amplified a DNA product from all 46 strains of subsp.avenaeoriginating from foxtail, oats, corn, rice, sugarcane, and millet; the type strains of subsp.cattleyaefrom orchid; and 11 strains of subsp.citrullifrom watermelon. Real-timeavenae-specific primers Oafl/Oar2 and probe AaP1 reacted with all 40 strains of subsp.avenae, the type strain of subsp.cattleyae, and 8 strains of subsp.citrulli. Classicalcitrulli-specific primers Aacf2/Aacr2 and real-time primers Aacf3/Aacr2 and probe AaP2 amplified a PCR product from all 11 strains of subsp.citrulli, but not from any other strain of subsp.avenae. None of over 50 other bacteria tested generated a PCR product with either classical primers or real-time primers and probe. The species-specific classical primers Oafl/Oarl and real-time primers Oafl/Oar2 and probe AaP1 should prove useful for rapid identification ofA. avenaestrains from all hosts, whereas thecitrulli-specific classical primers Aacf2/Aacr2 and real-time primers Aacf3/Aacr2 and probe AaP2 should be useful for specific identification of subsp.citrulli.