Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis

• Published in: Diagnostic microbiology and infectious disease 1999-03, Vol.33 (3), p.163-171
• Main Authors: Englund, Stina, Ballagi-Pordány, András, Bölske, Göran, Johansson, Karl-Erik
• Format: Article
• Language: English
• Subjects: Animal bacterial diseases; Animals; Bacterial diseases; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - microbiology; Culture Media; DNA Transposable Elements; Feces - microbiology; Fundamental and applied biological sciences. Psychology; Ileum - microbiology; Infectious diseases; Lymph Nodes - microbiology; Medical sciences; Microbiology; Molecular Mimicry; Mycobacterium avium subsp. paratuberculosis - genetics; Mycobacterium avium subsp. paratuberculosis - growth & development; Mycobacterium avium subsp. paratuberculosis - isolation & purification; Paratuberculosis - diagnosis; Paratuberculosis - microbiology; Polymerase Chain Reaction - methods; Sensitivity and Specificity
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/S0732-8893(98)00098-4

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne’s disease in ruminants. The current methods for detection of M. avium subsp. paratuberculosis are slow and insensitive. We report the use of a polymerase chain reaction (PCR) based on IS 900 to confirm growth of M. avium subsp. paratuberculosis in primary bacterial cultures from bovine tissue and fecal samples. The use of PCR on single colonies reduced the time for analysis by 2 months compared with conventional methods. We also report the development of a nested PCR based on IS 900 and the development of a positive internal control molecule, a so-called mimic. The system was tested with spiked tissue samples, and the sensitivity was estimated to 10 CFU per sample. Seventeen tissue samples, previously found M. avium subsp. paratuberculosis positive by microbiological culture, were analyzed by nested PCR and the efficiency of the PCR was checked by co-amplification of the mimic. Absence of the mimic amplicon indicated inhibition of the amplification. Ten of the samples were positive and five were negative, as judged from the presence or absence of the IS 900 PCR product. Two negative samples could not be judged because of inhibition revealed by mimic molecules. It was concluded that the nested PCR, together with the mimic, could be a useful tool in screening tissue materials.