A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsilar swabs specimens of pigs
Autor: | E.M. Kamp, T.M.M. Vermeulen, H.E.C.M. Buys, M.A. Smith, M.F. de Jong, F.H. Reek, G.C.A.M. Bokken |
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Jazyk: | angličtina |
Rok vydání: | 1996 |
Předmět: |
0301 basic medicine
DNA Bacterial food.ingredient Pasteurella multocida 040301 veterinary sciences Swine 030106 microbiology Palatine Tonsil Pasteurella Infections Enzyme-Linked Immunosorbent Assay medicine.disease_cause Polymerase Chain Reaction Sensitivity and Specificity Microbiology law.invention 0403 veterinary science 03 medical and health sciences Microtiter plate food law otorhinolaryngologic diseases medicine Life Science Agar Animals Polymerase chain reaction Swine Diseases General Veterinary biology Toxin Hybridization probe 04 agricultural and veterinary sciences biology.organism_classification Isolation (microbiology) Molecular biology Nasal Mucosa ID-Lelystad Instituut voor Dierhouderij en Diergezondheid ID Lelystad Institute for Animal Science and Health Primer (molecular biology) |
Zdroj: | Journal of Veterinary Diagnostic Investigation, 8, 304-309 Journal of Veterinary Diagnostic Investigation 8 (1996) |
ISSN: | 1040-6387 |
Popis: | A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin-of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific. |
Databáze: | OpenAIRE |
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