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Cost‐effective optimization of real‐time PCR ‐based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases
Author(s) -
Fachmann M.S.R.,
Josefsen M.H.,
Hoorfar J.,
Nielsen M.T.,
Löfström C.
Publication year - 2015
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/jam.12937
Subject(s) - polymerase chain reaction , salmonella , campylobacter , real time polymerase chain reaction , biology , detection limit , dna , microbiology and biotechnology , dna extraction , chromatography , bacteria , chemistry , genetics , gene
Abstract Aims The aim of this study was to cost‐effectively improve detection of foodborne pathogens in PCR inhibitory samples through the use of alternative DNA polymerases. Methods and Results Commercially available polymerases ( n = 16) and PCR master mixes ( n = 4) were screened on DNA purified from bacterial cells in two validated real‐time PCR assays for Campylobacter and Salmonella . The five best performing (based on: limit of detection ( LOD ), maximum fluorescence, shape of amplification curves and amplification efficiency) were subsequently applied to meat and faecal samples. The VeriQuest qPCR master mix performed best for both meat and faecal samples ( LOD s of 10 2 and 10 4 CFU ml −1 in the purest and crudest DNA extractions respectively) compared with Tth ( LOD = 10 2 –10 3 and 10 5 –10 6 CFU ml −1 ). AmpliTaqGold and HotMasterTaq both performed well ( LOD = 10 2 –10 4 CFU ml −1 ) with meat samples and poorly ( LOD = 10 3 –10 6 CFU ml −1 /not detected) with faecal samples. Conclusions Applying the VeriQuest qPCR master mix in the two tested real‐time PCR assays could allow for simpler sample preparation and thus a reduction in cost. Significance and Impact of the Study This work exemplifies a cost‐effective strategy for optimizing real‐time PCR ‐based assays. However, a DNA polymerase suitable for one assay and sample type is not necessarily optimal for other assays or sample types.