Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR
Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR
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开发用于 PCR 特异性检测大肠杆菌 O157 的 O 抗原生物合成基因引物
DOI：
10.1128/aem.65.7.2954-2960.1999
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发表时间：
1999
影响因子：
4.4
通讯作者：
Margie D. Lee
中科院分区：
文献类型：
--
作者：
J. Maurer;D. Schmidt;P. Petrosko;S. Sánchez;Lance Bolton;Margie D. Lee
摘要 革兰氏阴性细菌中每个 O 抗原亚基的化学成分反映了每个 rfb 操纵子内独特的 DNA 序列。通过表征每个 rfb 操纵子所含的 DNA 序列，可以生成通常与细菌感染相关的大肠杆菌 O 血清型的诊断血清型特异性探针。最近，从大肠杆菌 O157:H7 粘粒文库中，用 O157 特异性抗血清鉴定出 O 抗原阳性粘粒。通过使用粘粒DNA作为探针，通过Southern分析鉴定了大肠杆菌O157血清型特有的几个DNA片段。其中一些 DNA 片段是从 O157 抗原阳性粘粒中亚克隆的，并在 Southern 分析中用作 DNA 探针。通过 Southern 分析鉴定出质粒 pDS306 内的一个 DNA 片段，该片段对大肠杆菌 O157 血清型具有特异性。该质粒的 DNA 序列揭示了与两个 rfb 基因的同源性，其中第一个基因编码 GDP-甘露糖脱水酶。这些 rfb 基因与霍乱弧菌和小肠结肠炎耶尔森氏菌血清型 O:8 中的 O 抗原生物合成基因相似。寡核苷酸引物对设计用于扩增大肠杆菌 O157 血清型的 420 bp DNA 片段。 PCR 测试针对大肠杆菌 O157 血清型。 PCR 使用 O157 特异性 rfb 寡核苷酸引物检测到了少至 10 个细胞。结合当前的富集方案，通过 PCR 进行 O157 血清分型将为鉴定大肠杆菌 O157 提供快速、特异且灵敏的方法。
ABSTRACT The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within eachrfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe toEscherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coliO157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.