title: Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine.
abstract: A number of polymerase chain reaction (PCR)-based diagnostic tests have been developed for Mycoplasma hyopneumoniae, including one from this research group. This report presents further development, optimization, and standardization of a nested PCR test. Detection sensitivity was 1 fg of M. hyopneumoniae chromosomal DNA (approximately 1 organism). This exceeded the sensitivity of or compared favorably with other published PCR tests. Polymerase chain reaction primers to porcine beta2-microglobulin were included as internal controls for amplifiable chromosomal DNA from porcine samples. To standardize the test, a number of samples from experimentally infected pigs, including nasal, tonsil, tracheobronchial swabs, lung tissue, bronchial alveolar lavage (BAL) fluid, and tracheobronchial brush samples, were examined by PCR. Samples obtained from BAL fluid and tracheobronchial sites were most predictive of infection, whereas nasal swabs and lung tissue were not reliable indicators of experimentally induced infection. In conclusion, the nested PCR developed for this study was found to be a highly sensitive and specific diagnostic tool for M. hyopneumoniae, but the enhanced sensitivity may be unnecessary if the proper sites are sampled.
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Veterinary Medical Research Institute, Iowa State University, Ames 50011, USA.
authors.forename: Kathy T
authors.initials: KT
authors.lastname: Kurth
authors.forename: Tsungda
authors.initials: T
authors.lastname: Hsu
authors.forename: Eric R
authors.initials: ER
authors.lastname: Snook
authors.forename: Eileen L
authors.initials: EL
authors.lastname: Thacker
authors.forename: Brad J
authors.initials: BJ
authors.lastname: Thacker
authors.forename: F Chris
authors.initials: FC
authors.lastname: Minion
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics* / Q000472:pathogenicity; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000235:genetics / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012016:Reference Values; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004279:DNA, Viral