Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours.
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.
Stanley
Emma C
EC
Division of Food Sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Sutton Bonington LE12 5RD, United Kingdom. cath.rees@nottingham.ac.uk
Mole
Richard J
RJ
Smith
Rebecca J
RJ
Glenn
Sarah M
SM
Barer
Michael R
MR
McGowan
Michael
M
Rees
Catherine E D
CE
D000818:Animals; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000235:genetics; D005516:Food Microbiology; D006801:Humans; D008892:Milk / Q000382:microbiology; D009160:Mycobacteriophages / Q000254:growth & development*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification* / Q000821:virology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay
D004269:DNA, Bacterial