Factors affecting the relationship between quantitative polymerase chain reaction (qPCR) and culture‐based enumeration of Enterococcus in environmental waters

• Published in: Journal of applied microbiology 2014-03, Vol.116 (3), p.737-746
• Main Authors: Raith, M.R, Ebentier, D.L, Cao, Y, Griffith, J.F, Weisberg, S.B
• Format: Article
• Language: English
• Subjects: Bathing Beaches; Biological and medical sciences; Cell Culture Techniques; DNA; Enterococcus; Enterococcus - genetics; Enterococcus - isolation & purification; Enterolert; Fundamental and applied biological sciences. Psychology; membrane filtration; Microbiology; monitoring; Polymerase Chain Reaction; qPCR; quantitative polymerase chain reaction; recreational water quality; regression analysis; Scorpiones; Seawater - microbiology; target difference; Water Microbiology; Water Quality
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.12383

AIMS: To determine the extent to which discrepancies between qPCR and culture‐based results in beach water quality monitoring can be attributed to: (i) within‐method variability, (ii) between‐method difference within each method class (qPCR or culture) and (iii) between‐class difference. METHODS AND RESULTS: We analysed 306 samples using two culture‐based (EPA1600 and Enterolert) and two qPCR (Taqman and Scorpion) methods, each in duplicate. Both qPCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log₁₀unit overestimation by qPCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between‐replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within‐method and within‐class variability, 8% of the samples exhibited true between‐class discrepancy where both qPCR methods indicated beach posting while both culture methods did not. CONCLUSION: Measurement target difference (DNA vs growth) accounted for the majority of the qPCR‐vs‐culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to quantify the frequency with which culture‐vs‐qPCR discrepancies can be attributed to target difference ‐ vs ‐ method variability.