Evaluation of 5′ Nuclease Assay for Detection of Actinobacillus pleuropneumoniae

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Abstract
Sequence detection by the 5′ nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae , which gave an amplicon of 92 bp. The test was evaluated with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a C t value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5′ nuclease assay represents a test with 100% sensitivity and 100% specificity. A high degree of reproducibility of the test was demonstrated. If samples with C t values of ≤30 are considered positive, the detection limit of the assay was 1 CFU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30, nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of A. pleuropneumoniae , as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with A. pleuropneumoniae , is the same level as that of a PCR test based on the omlA gene described previously. The 5′ nuclease assay represents a fast method for species-specific detection and identification of A. pleuropneumoniae in pure and mixed cultures. The evaluation shows, however, that a C t value cutoff limit of ≤30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.