Molecular cloning and sequencing of the aroA gene from Actinobacillus pleuropneumoniae and its use in a PCR assay for rapid identification.
The gene (aroA) of Actinobacillus pleuropneumoniae, serotype 2, encoding 5-enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. A pair of primers from the 5' and 3' termini were selected to be the basis for development of a specific PCR assay. A DNA fragment of 1,025 bp was amplified from lysed A. pleuropneumoniae serotypes 1 to 12 of biovar 1 or from isolated DNA. No PCR products were detected when chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified when Actinobacillus equuli chromosomal DNA was used as a target, which could be easily differentiated by its NAD independence. The PCR assay developed was very sensitive, with lower detection limits of 12 CFU with A. pleuropneumoniae cells and 0.8 pg with extracted DNA. Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis of A. pleuropneumoniae infections.
Hernanz Moral
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C
Departamento de Sanidad Animal, Microbiología e Inmunología, Facultad de Veterinaria, Universidad de León, 24071 León, Spain.
Cascón Soriano
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A
Sánchez Salazar
M
M
Yugueros Marcos
J
J
Suárez Ramos
S
S
Naharro Carrasco
G
G
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000302:isolation & purification; D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase