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

Fachmann, Mette Sofie Rousing; Josefsen, Mathilde Hartmann; Hoorfar, Jeffrey; Nielsen, Mette Thorup; Löfström, Charlotta

doi:10.1111/jam.12937

Cited by 8 publications

(6 citation statements)

References 40 publications

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“…The highly specific identification of pathogens is enabled by PCR in a few hours. However, Fachmann et al reported that the limit of detection (LOD) of real-time PCR-based detection of S. enterica was 10 3 CFUs·mL −1 [ 9 ], which is a higher concentration than that of the conventional culture method. In addition, it is necessary to reduce the amount of PCR reagents used to assay many samples because PCR reagents are expensive.…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device

et al. 2016

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Rapid detection of food-borne pathogens is essential to public health and the food industry. Although the conventional culture method is highly sensitive, it takes at least a few days to detect food-borne pathogens. Even though polymerase chain reaction (PCR) can detect food-borne pathogens in a few hours, it is more expensive and unsatisfactorily sensitive relative to the culture method. We have developed a method to rapidly detect Salmonella enterica by using a compact disc (CD)-shaped device that can reduce reagent consumption in conventional PCR. The detection method, which combines culture and PCR, is more rapid than the conventional culture method and is more sensitive and cheaper than PCR. In this study, we also examined a sample preparation method that involved collecting bacterial cells from food. The bacteria collected from chicken meat spiked with S. enterica were mixed with PCR reagents, and PCR was performed on the device. At a low concentration of S. enterica, the collected S. enterica was cultured before PCR for sensitive detection. After cultivation for 4 h, S. enterica at 1.7 × 104 colony-forming units (CFUs)·g−1 was detected within 8 h, which included the time needed for sample preparation and detection. Furthermore, the detection of 30 CFUs·g−1 of S. enterica was possible within 12 h including 8 h for cultivation.

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Section: Introductionmentioning

confidence: 99%

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device

et al. 2016

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“…These samples are challenging for rapid PCR-based detection, as certain PCR inhibitors are expected and have to be removed prior to detection, e.g., heme from blood (23), myoglobin from tissue (24), and fat (25). PCR inhibition can be relieved by using a DNA polymerase more tolerant to inhibitors (26) or by adding PCR facilitators to the PCR master mix (27), but often a combination with a sample preparation step is needed in order to generate a PCR-compatible sample. Studies have shown that PCR inhibitors can be reduced by physical, biochemical, immunological, and physiological sample preparation methods.…”

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confidence: 99%

Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

Fachmann

Löfström

Hoorfar

et al. 2017

Appl Environ Microbiol

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Salmonella is recognized as one of the most important foodborne bacteria and has wide health and socioeconomic impacts worldwide. Fresh pork meat is one of the main sources of Salmonella, and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include Ͼ16 h of culture enrichment, in a few cases Ͻ12 h, thus requiring at least two working shifts. Here, we report a rapid (Ͻ5 h) and high-throughput method for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water and a real-time PCR-compatible sample preparation method based on filtration, centrifugation, and enzymatic digestion, followed by fast-cycling real-time PCR detection. The method was validated in an unpaired comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n ϭ 140) were either artificially contaminated with Salmonella at 0, 1 to 10, or 10 to 100 CFU/25 g of meat or naturally contaminated. Cohen's kappa for the degree of agreement between the rapid method and the reference was 0.64, and the relative accuracy, sensitivity, and specificity for the rapid method were 81.4, 95.1, and 97.9%, respectively. The 50% limit of detections (LOD 50 s) were 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low-risk meat, providing savings for meat producers, and it will help contribute to improved food safety.IMPORTANCE While the cost of analysis and hands-on time of the presented rapid method were comparable to those of reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage, but consumers and retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in the detection of other pathogenic bacteria in different sample types.KEYWORDS Salmonella, food safety, foodborne pathogens, fresh meat, rapid tests, real-time PCR, sample preparation, validation S almonella is a global foodborne pathogen responsible for approximately 30% of foodborne outbreaks in the United States and 23% in the European Union (1, 2). Pork meat remains an important source of human salmonellosis, which is why the absence of Salmonella is confirmed at critical control points at abattoirs (2). However, the reference culture-based methods require several days to obtain a test result (3, 4), thereby postponing the market release of fresh meat.

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“…and, of course, combinations of both can be adapted. As with Fachmann and colleagues [ 19 ], we originally desired solely a cost-effective polymerase exchange without performing too many optimizations other than specific necessities such as the initial denaturation of the AmpliTaq Gold. However, this extended denaturation step appears to be insufficient for satisfactory amplification and the performance of most other polymerases was also poor ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Buzard and colleagues compared ten different qPCR mastermixes with four bacterial assays and demonstrated differences between the mastermixes and assays [ 29 ]. Additionally, Fachmann et al, for cost-effective qPCR optimization, compared 16 polymerases with two bacterial assays and different sample types and concluded that the choice of the appropriate polymerase depends upon the assay, question and sample type [ 19 ]. Likewise, unequal performances of diverse assays using different mastermixes were demonstrated recently [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

Witte

Sickha

Mester

et al. 2018

Biomolecular Detection and Quantification

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The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.

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

Abstract: 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.

Cited by 8 publications

References 40 publications

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device

Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

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