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Long‐amplicon propidium monoazide‐ PCR enumeration assay to detect viable C ampylobacter and S almonella
Author(s) -
Banihashemi A.,
Dyke M.I.,
Huck P.M.
Publication year - 2012
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2012.05382.x
Subject(s) - propidium monoazide , amplicon , salmonella enterica , biology , campylobacter jejuni , campylobacter , microbiology and biotechnology , salmonella , enumeration , polymerase chain reaction , real time polymerase chain reaction , bacteria , gene , biochemistry , genetics , mathematics , combinatorics
Aims The effect of amplicon length on the ability of propidium monoazide‐ PCR ( PMA ‐ PCR ) to reliably quantify viable cells without interference from dead cells was tested on heat‐ and ultraviolet ( UV )‐killed S almonella enterica and C ampylobacter jejuni , two important enteric pathogens of concern in environmental, food and clinical samples. Methods and Results PMA treatment followed by quantitative PCR (q PCR ) amplification of short DNA fragments (<200 bp) resulted in incomplete signal inhibition of heat‐treated S alm. enterica (3 log reduction) and C amp. jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6 kb) completely suppressed the dead cell signal. PMA pretreatment of UV ‐irradiated cells did not affect PCR amplification, but long‐amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA . Conclusions The long‐amplicon PMA ‐ PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including S almonella and C ampylobacter that are difficult to enumerate using culture‐based procedures. Significance and Impact of the Study PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane‐damaged cells, and also for inactivation mechanisms that cause direct DNA damage.