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Journal of Clinical Microbiology
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Clinical Veterinary Microbiology

Molecular Cloning and Sequencing of thearoA Gene from Actinobacillus pleuropneumoniaeand Its Use in a PCR Assay for Rapid Identification

Carmen Hernanz Moral, Alberto Cascón Soriano, María Sánchez Salazar, Javier Yugueros Marcos, Susana Suárez Ramos, German Naharro Carrasco
Carmen Hernanz Moral
Departamento de Sanidad Animal, Microbiologı́a e Inmunologı́a, Facultad de Veterinaria, Universidad de León, 24071 León, Spain
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Alberto Cascón Soriano
Departamento de Sanidad Animal, Microbiologı́a e Inmunologı́a, Facultad de Veterinaria, Universidad de León, 24071 León, Spain
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María Sánchez Salazar
Departamento de Sanidad Animal, Microbiologı́a e Inmunologı́a, Facultad de Veterinaria, Universidad de León, 24071 León, Spain
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Javier Yugueros Marcos
Departamento de Sanidad Animal, Microbiologı́a e Inmunologı́a, Facultad de Veterinaria, Universidad de León, 24071 León, Spain
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Susana Suárez Ramos
Departamento de Sanidad Animal, Microbiologı́a e Inmunologı́a, Facultad de Veterinaria, Universidad de León, 24071 León, Spain
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German Naharro Carrasco
Departamento de Sanidad Animal, Microbiologı́a e Inmunologı́a, Facultad de Veterinaria, Universidad de León, 24071 León, Spain
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DOI: 10.1128/JCM.37.5.1575-1578.1999
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    Fig. 1.

    Restriction endonuclease map of the aroAlocus obtained with plasmid pAP2. Solid boxes represent A. pleuropneumoniae-cloned DNA. The thicker box is the A. pleuropneumoniae aroA gene, which was oriented from 5′ (left) to 3′ (right). The thin line represents pUC18 plasmid DNA.

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    Fig. 2.

    Nucleotide sequence of the aroA gene and the amino acid sequence deduced from the open reading frame of thearoA gene. DNA bases (top line) and amino acids (one-letter code) (below) are shown, and nucleotides are numbered to the right of the sequences. The ATG initiation code (boldface) is preceded by a potential Shine-Dalgarno sequence (double underlining). Primers which have been used for PCR analysis are underlined. The dash indicates the TGA termination codon (boldface).

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    Fig. 3.

    CLUSTAL computer alignment of the deduced amino acid sequences encoded by the aroA gene from A. pleuropneumoniae (APL), H. influenzae (HINFL), P. haemolytica (PHAEM), Yersinia pestis (YERPES),Salmonella typhimurium (SALTYPHI), E. coli(ECOLI), K. pneumoniae (KLEBPNEU), Aeromonas salmonicida (ASA), and A. hydrophila (AHY). Identical amino acids in all species are indicated by an asterisk; conservative substitutions are indicated by a dot.

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    Fig. 4.

    Agarose gel electrophoresis of PCR amplification products from total DNA from E. coli (lane 1), A. pleuropneumoniae serotypes 1 to 12 (ATCC 27088, ATCC 27089, ATCC 27090, NCTC 11384, NCTC 11383, ATCC 33590, WF 83, 405, CVJ 13261, D 13039, 16153, and 8329, respectively) (lanes 2 to 13), A. equuli NCTC 8529 (lane 14), H. influenzae ATCC 35056 (lane 15), P. haemolytica ATCC 33396 (lane 16), P. multocida ATCC 12048 (lane 17), K. pneumoniae (patient isolate) (lane 18), and A. hydrophila SO2/2 (lane 19). M, standard DNA size markers (HaeIII-digested φX174).

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    Fig. 5.

    Agarose gel electrophoresis of fragments produced bySau3A digestion of A. pleuropneumoniae aroA genes amplified by PCR. Lanes 1 to 12, serotypes 1 to 12; lane 13, A. equuli. M, standard DNA size markers (HaeIII-digested φX174).

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Molecular Cloning and Sequencing of thearoA Gene from Actinobacillus pleuropneumoniaeand Its Use in a PCR Assay for Rapid Identification
Carmen Hernanz Moral, Alberto Cascón Soriano, María Sánchez Salazar, Javier Yugueros Marcos, Susana Suárez Ramos, German Naharro Carrasco
Journal of Clinical Microbiology May 1999, 37 (5) 1575-1578; DOI: 10.1128/JCM.37.5.1575-1578.1999

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Molecular Cloning and Sequencing of thearoA Gene from Actinobacillus pleuropneumoniaeand Its Use in a PCR Assay for Rapid Identification
Carmen Hernanz Moral, Alberto Cascón Soriano, María Sánchez Salazar, Javier Yugueros Marcos, Susana Suárez Ramos, German Naharro Carrasco
Journal of Clinical Microbiology May 1999, 37 (5) 1575-1578; DOI: 10.1128/JCM.37.5.1575-1578.1999
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KEYWORDS

Actinobacillus pleuropneumoniae
Alkyl and Aryl Transferases
polymerase chain reaction

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