Optimization of a 12-Hour TaqMan PCR-Based Method for Detection of
            <i>Salmonella</i>
            Bacteria in Meat

Josefsen, Mathilde Hartmann; Krause, Michael; Hansen, Flemming; Hoorfar, Jeffrey

doi:10.1128/aem.02823-06

Cited by 88 publications

(64 citation statements)

References 27 publications

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“…and S. Enteritidis in environmental samples. VI 1,6,14,25:a:e,n,z15 (CFSAN015097). The DNA extracts from 12 control organisms of a Salmonella panel were prepared from 1.0 ml of overnight culture using InstaGene matrix, according to the manufacturer's instructions (catalog no.…”

Section: Figmentioning

confidence: 99%

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

Kasturi

Drgon

2017

Appl Environ Microbiol

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The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA-and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S. Enteritidis isolates and 100% exclusivity for the 297 nonEnteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method.IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples. KEYWORDS S. Enteritidis, environmental samples, Salmonella, validation of PCR method, real-time PCR Salmonella is one of the major causes of foodborne illnesses, with the U.S. Centers for Disease Control and Prevention (CDC) estimating that ϳ1.2 million cases of salmonellosis occur annually in the United States alone. Salmonella infections cause severe illness in infants, the elderly, and immunocompromised individuals. Most infections in humans result from eating contaminated food or drinking contaminated water. Sources of Salmonella infections include foods of animal origin, dairy products, pet food, fresh produce, and foods contaminated during processing. Moreover, Salmonella outbreaks following the consumption of eggs contaminated with Salmonella enterica serovar Enteritidis are relatively common (1).The expeditious detection of Salmonella species in food and environmental samples requires rapid, efficient, and validated methods. A number of conventional and realtime PCR methods have been reported for the detection of Salmonella in naturally or artificially contaminated food samples (2-9) and fecal samples (10). A few validation

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Section: Figmentioning

confidence: 99%

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

Kasturi

Drgon

2017

Appl Environ Microbiol

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“…In addition, as Salmonella in meat is often injured from the chilling process, the international standards recommend a two-step enrichment procedure, where the first nonselective preenrichment step is followed by a selective enrichment step. Several attempts to reduce the enrichment time have been reported (13)(14)(15)(16). Studies in which shortening of the 16-to 20-h reference enrichment time was investigated reported that at least 8 h of incubation was required for meat samples (15,16), while a 3-h incubation resulted in the detection of Salmonella in highly contaminated oysters (17).…”

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confidence: 99%

“…Several attempts to reduce the enrichment time have been reported (13)(14)(15)(16). Studies in which shortening of the 16-to 20-h reference enrichment time was investigated reported that at least 8 h of incubation was required for meat samples (15,16), while a 3-h incubation resulted in the detection of Salmonella in highly contaminated oysters (17). Omitting the time-consuming enrich-ment step would therefore be optimal, and several direct detection methods have also been published (9,(18)(19)(20).…”

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confidence: 99%

“…Studies have shown that PCR inhibitors can be reduced by physical, biochemical, immunological, and physiological sample preparation methods. These include flotation (18), filtration (9,20), adsorption and addition of surfactants (19), enzymatic digestion (9), DNA extraction (28), and enrichment in a PCR-compatible medium (15), all of which were considered when designing the described rapid method.…”

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confidence: 99%

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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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“…An additional centrifugation step just prior to the PCR reaction was suggested by Chen et al (1997) to better remove PCR inhibitors. Wilson (1997) recommended a washing step to reduce the fat and other residues in foods which could inhibit the PCR reaction, whereas Josefsen et al (2007) reported lower reproducibility with the washing step. Immunomagnetic separation (IMS) with an additional enrichment step was used to overcome false-negative results of Salmonella in chicken products (Soumet et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

Patel

Bhagwat

2008

Acta Veterinaria Hungarica

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A real-time PCR assay was evaluated for the rapid detection (10 h) of Salmonella in meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated with Salmonella enterica serovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

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Optimization of a 12-Hour TaqMan PCR-Based Method for Detection of Salmonella Bacteria in Meat

Cited by 88 publications

References 27 publications

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

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

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

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