PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely r...
PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3''''-terminus of the species-specific apxIVA gene and already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A pleuropnuemoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of 10 strains representing 8 different species bacteria including species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR was evaluated on mixed bacterial cultures of nasal and tonsillar swabs, as well as lung lesions from disease pigs. Positive results were obtained from 9 of 50 (18%), 19 of 50(38%), and 10 of 50(20%) mixed bacterial cultures of tonsillar swabs, nasal swabs, and necrotic lung lesions, respectively. The results indicate that the multiplex PCR assay is a sensitive, highly specific and effective diagnostic tool for identification and detection of A. pleuropneumoniae.
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