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Identification and Detection of Actinobacillus pleuropneumoniae in Infected and Subclinically Infected pigs by Multiplex PCR Based on the Genes ApxIVA and OmlA

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Agricultural Sciences in China 2006年第2期5卷 146-154页

作者： XIAO Guo-sheng CAO San-jie DUAN Li-li WEN Xin-tian MA Xiao-ping CHEN Hua-mei Laboratory of Animal Infectious Disease and Microarray/Key Laboratory of Animal Disease and Human Health of Sichuan Province College of Animal Science and Veterinary Medicine Sichuan Agricultural University Ya＇an 625014 P.R.China

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 apxlVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 （26%） of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.

关键词： multiplex PCR Actinobacillus pleuropneumoniae pig bacteria apxIVA and omlA genes

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