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Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

Division of Food Sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Sutton Bonington LE12 5RD, United Kingdom,¹ Biotec Laboratories, 32 Anson Road, Marltesham Heath, Ipswich IP5 3RG, United Kingdom,² Department of Infection, Immunity and Inflammation, University of Leicester, Medical Sciences Building, PO Box 138, University Rd., Leicester LE1 9HN, United Kingdom,³ The University of Queensland, School of Veterinary Science, Brisbane, Queensland, Australia⁴

Received 21 July 2006/ Accepted 28 December 2006

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.

Published ahead of print on 26 January 2007.

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• Altic, L. C., Rowe, M. T., Grant, I. R. (2007). UV Light Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk as Assessed by FASTPlaqueTB Phage Assay and Culture. Appl. Environ. Microbiol. 73: 3728-3733 [Abstract] [Full Text]