Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable campylobacter cells.
The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R(2)) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10(4)) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.
Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
Diana
D
Seinige
Carsten
C
Krischek
Günter
G
Klein
Corinna
C
Kehrenberg
D000818:Animals; D001386:Azides / Q000378:metabolism*; D058491:Bacterial Load / Q000379:methods*; D017000:Campylobacter coli / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D016123:Campylobacter jejuni / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D002470:Cell Survival; D002645:Chickens; D004791:Enzyme Inhibitors / Q000378:metabolism; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D001386:Azides; D004791:Enzyme Inhibitors; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium