Cost-effective optimization of real-time PCR-based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases

• 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 (LODs of 102 and 104 CFU ml-1 in the purest and crudest DNA extractions respectively) compared with Tth (LOD = 102-103 and 105-106 CFU ml-1). AmpliTaqGold and HotMasterTaq both performed well (LOD = 102-104 CFU ml-1) with meat samples and poorly (LOD = 103-106 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.

Nyckelord

Salmonella

Campylobacter

Detection

DNA polymerase

Faeces

Inhibitors

Minced meat

Sample preparation

locked nucleic acid

Taq polymerase

bacterial DNA

DNA directed DNA polymerase

bacterium

cost analysis

detection method

DNA

enzyme activity

feces

inhibition

inhibitor

meat

optimization

polymerase chain reaction

real time

Article

bacterial cell

bacterium culture

bacterium detection

colony forming unit

controlled study

cost effectiveness analysis

DNA extraction

feces analysis

food contamination

limit of detection

nonhuman

real time polymerase chain reaction

Salmonella enterica serovar Typhimurium

animal

chemistry

economics

evaluation study

genetics

isolation and purification

metabolism

microbiology

procedures

Bacteria (microorganisms)

Animals

DNA

Bacterial

DNA-Directed DNA Polymerase

Real-Time Polymerase Chain Reaction

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