Multiplex TaqMan Real-Time PCR (qPCR) Assay Targeting prot6E and invA Genes for Fast and Accurate Detection of Salmonella Enteritidis

González-Escalona, Narjol; Zhang, Guodong; Brown, Eric W.

doi:10.5772/28657

Cited by 2 publications

(1 citation statement)

References 36 publications

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“…All other serovars of group D were positive with both of the prt and tyv PCR reagents but not with the Sdf-1 reagent. Thus, the new method is more reliable than current methods: the protE6-specific PCR reagent recog-nized several phage types of S. Enteritidis but not all (9,32), and the Sdf-1-specific reagent (33,34) would have identified the S. Montevideo isolate as S. Enteritidis.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

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

Kasturi

Drgon

2017

Appl Environ Microbiol

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Technical note: Development of a feed matrix as inoculum in Salmonella infection studies in piglets

Litjens

Oudshoorn

Hil

2017

Journal of Animal Science

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Current methods used for oral administration of pathogens to piglets are stressful for the animals because of fixation and use of inoculation methods such as oral gavage. In the present study an alternative way to challenge piglets with Typhimurium (ST) is investigated. The strategy was to incorporate the in a feed matrix, which is fed to the piglets and eaten voluntary. Different types of feed matrices were tested for their absorption capacity, handling properties, and palatability to piglets. Furthermore, the viability of ST in feed matrices was studied. The ST could be incorporated in ladyfinger biscuits; a 2-cm piece absorbs 1 mL of culture media. The ladyfinger biscuits were very well accepted by the piglets. After a short training period, the piglets consumed the -incorporated biscuits out of the feeding trough. In this way the animals were infected in a more natural way and without stress. The safety of farm workers was also increased because the incorporated ST results in less spoilage and spreading of ST during the infections. Results indicated that the ST cell count was reduced by only 0.2 log unit to 8.7 log cfu per inoculum after 24 h at 4°C incorporation of ST into the biscuits, which is sufficient for an infection study and indicates excellent viability of the . The viability was also indicated by increased fecal shedding of ST. Thus, it is concluded that ladyfinger biscuits are a suitable matrix to challenge piglets with ST.

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Multiplex TaqMan Real-Time PCR (qPCR) Assay Targeting prot6E and invA Genes for Fast and Accurate Detection of Salmonella Enteritidis

Cited by 2 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

Technical note: Development of a feed matrix as inoculum in Salmonella infection studies in piglets

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