Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

Anderson, A. B.; Pietsch, Klaus; Zucker, R.; Mayr, A.; Müller-Hohe, Elke; Messelhäußer, Ute; Sing, Andreas; Busch, Ulrich; Huber, Ingrid

doi:10.1007/s12161-010-9142-8

Cited by 79 publications

(51 citation statements)

References 36 publications

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“…A few validation studies have been reported for food samples that were spiked with Salmonella (8,9,11,12,42), but no validation studies have been reported for environmental samples. The primer sets that have been used for the amplification of Salmonella DNA by different methods differed in their detection limits, lacked disclosure of the internal amplification control (IAC) DNA sequence used (8,9,(11)(12)(13)(14), or did not include a wide range of epidemiologically important isolates. In addition, the matrices and levels of contaminating competing organisms present in each environmental sample appeared to be unique, making the validation of methods for screening environmental samples a challenge.…”

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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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confidence: 99%

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

Kasturi

Drgon

2017

Appl Environ Microbiol

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“…Before artificial contamination, the presence of Salmonella spp. in samples was excluded by testing with the cultural method according to SRPS EN ISO 6579:2008 (ISO, ) and real‐time PCR method (Anderson et al, ). Then, 25 g of each artificially contaminated samples were mixed with 225 ml buffered peptone water (BPW) (Oxoid, UK), homogenized and incubated in accordance with SRPS EN ISO 6579:2008 (ISO, ).…”

Section: Methodsmentioning

confidence: 99%

“…Several real‐time PCR protocols were developed with the aim of detecting Salmonella spp. in food (Anderson et al, ; Josefsen, Krause, Hansen, & Hoorfar, ; Krämer et al, ; Malorny et al, ; Rodriguez‐Lazaro, Hernandez, Esteve, Hoorfar, & Pla, ). Also, there are a number of available kits that apply real‐time PCR method for the detection of Salmonella (Assurance GDS for Salmonella— BioControl Systems, Foodproof Salmonella Detection Kit—BIOTECON Diagnostics, GeneDisc Salmonella —Pall Corp., iQ‐Check Salmonella II Kit—BioRad, MicroSEQ Salmonella spp.…”

Section: Introductionmentioning

confidence: 99%

“…The aims of this study were: Optimization and in‐house validation of modified real‐time PCR protocol for detecting the inv A gene (Anderson et al, ) and comparing its efficiency with the standard method for Salmonella spp. detection in food (SRPS EN ISO 6579:2008). Optimization and in‐house validation of real‐time PCR protocol for detecting the ttr gene Salmonella spp.…”

Section: Introductionmentioning

confidence: 99%

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In‐house validation of real‐time PCR methods for detecting the INV A and TTR genes of Salmonella spp. in food

Dmitrić

Vidanović²,

Matović³

et al. 2017

J Food Process Preserv

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The microbiological standard method for the detection of Salmonella spp. is time‐consuming, and consequently there is a need for an alternative rapid methodology for its detection. In this study, two open‐formula real‐time PCR methods for detecting the inv A and ttr gene Salmonella spp. have been successfully optimized and in‐house validated. Different DNA extraction procedures were used (boiling, Chelex resin and standard I ‐ Biorad). The relative sensitivity and false positive ratio for the alternative methods were 100% and 0.0%, respectively. The relative trueness was in range from 96.8% to 99.2%. No false negative results were detected. The lowest Ct values were obtained using the protocol for detecting the ttr gene after DNA extraction by the Chelex. The results were compared in a large number of food and environmental samples to those of the SRPS EN ISO 6579:2008 standard method and commercial kit (IQ check® Salmonella II kit, Bio‐Rad, USA). Practical applications The whole procedure of real‐time PCR methods in this study was approximately 20 hr, in contrast to 4–5 days of analysis time for the standard method (SRPS EN ISO 6579:2008). The real‐time PCR method also provides a high level of sensitivity and specificity with a low risk of cross‐contamination. The implementation of the method for routine analysis will help improve safety in the food production chain, by providing results compatible with the ISO standard, but more rapidly.

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“…Moreover, relative sensitivity, specificity and accuracy (SE/SP/AC), positive and negative predictive values (PPV/NPV) and the index kappa of concordance (κ) were estimated from these data. Calculations of these parameters were performed as previously described [28,40,41].…”

Section: Evaluation Of the Specificity Accuracy And Precision Of Thementioning

confidence: 99%

Development, and complete evaluation, of a novel Most-Probable-Number (MPN) qPCR method for accurate and express quantification of Listeria monocytogenes in foodstuffs

Garrido‐Maestu

Vieites-Maneiro

Peñaranda

2015

Eur Food Res Technol

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Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

Cited by 79 publications

References 36 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

In‐house validation of real‐time PCR methods for detecting the INV A and TTR genes of Salmonella spp. in food

Development, and complete evaluation, of a novel Most-Probable-Number (MPN) qPCR method for accurate and express quantification of Listeria monocytogenes in foodstuffs

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