Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica

Hoorfar, Jeffrey; Ahrens, Peter; Rådström, Peter

doi:10.1128/jcm.38.9.3429-3435.2000

Cited by 231 publications

(84 citation statements)

References 25 publications

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“…The age of the animals was determined by the tooth eruption and replacement pattern and also by dental attrition (Boitani & Mattei, 1992). Identification of compatible colonies was performed suing the Phoenix 100 (Becton Dickinson) automated bacterial identification device and confirmed by detection of the invA gen by PCR (Hoorfar, Ahrens, & Rådström, 2000). PCR-confirmed isolates were sent to the National Reference Laboratory for Salmonella (Algete, Madrid, Spain)…”

Section: Postmortem Examinationmentioning

confidence: 99%

Outbreaks of antimicrobial resistantSalmonellaCholeraesuis in wild boars piglets from central‐western Spain

Molino

Risco²,

Blanco³

et al. 2018

Transbound Emerg Dis

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Salmonella enterica serovar Choleraesuis is the aetiological agent of swine paratyphoid being a highly invasive zoonotic pathogen. Wild boar natural populations are experiencing a demographical expansion as well as some farms are breeding this species to release for hunting with management sometimes identical to that of domestic pigs, including supplementation, grouping, and antibiotic treatments. This situation increases the chance of contact between wild boars and livestock, and potentially induces stress, with different sanitary consequences. The present work aims to describe the clinical features of recent outbreaks caused by S. Choleraesuis in wild boar from central-western Spain, as well as the antimicrobial resistance and phylogenetic relationships of isolates involved. 28 strains of S. Choleraesuis were isolated from 28 different wild boars belonging to 10 different game states located in central western Spain and submitted to the Clinical Veterinary Hospital (CVH) of the University of Extremadura. Samples were taken from different organs and cultured according to the ISO 6579:2002 procedure. Suspicious colonies were identified by PCR and antimicrobial resistance was evaluated by disc diffusion susceptibility test and the presence of the main resistance genes as well as 18 plasmid replicons frequently found among the Enterobacteriaceae was verified by PCR. Pulsed field gel electrophoresis was applied to determine the genetic relationship between isolates. The outbreaks under study were characterized by high mortality (35%-84%) and a septicaemic presentation. S. Choleraesuis was isolated from all the wild boars analysed, and 26 of the 28 isolates presented resistance to at least one antibiotic. The predominant resistances found were against sulphonamide, streptomycin, tetracycline, and doxicicline and sul1, strA-strB, and tetA were the most prevalent resistance genes among isolates. 10 strains carried FIIA, FIB+H/1 or FIIA+H/1 plasmids. PFGE classified the isolates into four different profiles, grouped into two clusters. This results show that prevention against S. Choleraesuis must be considered in the sanitary programs of the wild boar breeders.

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Section: Postmortem Examinationmentioning

confidence: 99%

Outbreaks of antimicrobial resistantSalmonellaCholeraesuis in wild boars piglets from central‐western Spain

Molino

Risco²,

Blanco³

et al. 2018

Transbound Emerg Dis

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show abstract

“…The primers for detecting E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica were targeted specifically on the rfbE (This study), hlyA (Wu et al 2004), nuc (Brakstad et al 1992) and invA genes (Hoorfar et al 2000), respectively. The rfbE gene encoding O157 lipopolysaccharide and for E. coli O157: H7 serogroup is unique (Oberst et al 1998), hlyA encoding listeriolysin for phagosomal escape into the host cell's cytosol (Wu et al 2004), the nuc (thermonuclease) gene in S. aureus (Brakstad et al 1992) and invA encoding an invasion protein in S. enterica (Hoorfar et al 2000). The primers were synthesized by Macrogen Company from South Korea.…”

Section: Primer Designmentioning

confidence: 99%

Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

et al. 2019

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Aims The aim of this study was to develop a multiplex touchdown PCR (multiplex TD‐PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. Methods and Results For this reason, a multiplex TD‐PCR assay with a pre‐enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis in pure culture and raw milk samples. The results showed that this protocol can eliminate the unwanted band or reduce significantly. The detection sensitivity of the single and multiplex TD‐PCR was one cell per ml in pure culture. Furthermore, the detection limit of multiplex TD‐PCR was one cell per 25 ml for artificially contaminated raw milk. We obtained similar results for detection of aforementioned pathogens in raw milk, after comparing the multiplex TD‐PCR method with the traditional culture, except in one or two samples. Conclusions Hence, the proposed multiplex TD‐PCR method could be confirmed as an effective way for rapid optimization of PCR reactions to increase specificity, sensitivity during gene amplification. Significance and Impact of the Study Hence, due to its simplicity, cost‐effectiveness and being time‐saving, it seems that this method is reasonable and economical for rapid optimization of PCR reactions.

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“…The surfaces of breast meat were then removed with a sterile scalpel to obtain an "internal part" of breast meat. Each prepared breast was divided into two 25-g samples: one was used to detect background levels of S. Typhimurium and L. monocytogenes using traditional standard methods (Bacteriological Analytical Manual of USFDA) and polymerase chain reaction (PCR) [detection of invA and hilA genes for Salmonella) (Hoorfar et al 2000;Mccabe et al 2011), and hlyA gene for L. monocytogenes (Amagliani et al 2010)] and the other was used for inoculation (Chen 2007). Briefly, 200 mL of prepared culture was spotted and spread on the upper meat surface (constant area) with a sterile glass rod and then the inoculated samples were kept at room temperature for 45 min to allow bacteria to attach (Hajmeer et al 2004;Chen 2007).…”

Section: Meat Sample Preparation and Inoculationmentioning

confidence: 99%

Optimization of an Acidified Sodium Chlorite Solution for Reducing Pathogenic Bacteria and Maintaining Sensory Characteristics of Poultry Meat in Simulation Slaughter Process

Wang

2012

Journal of Food Processing and Preservation

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Chicken breast meat was inoculated with Salmonella Typhimurium and Listeria monocytogenes and then washed with an acidified sodium chlorite (ASC) solution under conditions simulating those used in a commercial chicken processing plant. We evaluated the effects of ASC (concentrations ranging from 0 to 1 g/L, pH at 2.5, 3.5 and 6.5) on the reduction of microbial numbers, meat pH, color, total acceptability and chilled storage characteristics. The results showed that bacterial numbers were significantly reduced with increasing concentrations of ASC and with decreasing pH, reaching maximum reductions of 2.17 and 3.03 log cfu/g for S. Typhimurium and L. monocytogenes when washing with 1 g ASC/L at pH 2.5, respectively. Except for the treatment with 1 g ASC/L at pH 2.5, which resulted in a significant reduction of total acceptability of chicken meat, other treatments were all within an acceptable range. Considering the inactivation of bacteria and the changes in sensory characteristics of meat, we selected 0.8 g ASC/L at pH 2.5 as being optimal for our purpose. Practical Applications Pathogenic bacteria frequently occur in raw poultry meat. It is therefore essential to develop an effective measure for controlling pathogenic bacteria and maintaining sensory characteristics of poultry meat during the slaughter process. In this study, we investigated the efficacy of acidified sodium chlorite (ASC) solution, which has been suggested as an effective alternative to sodium hypochlorite, at various concentrations and acidity, in reducing pathogenic microbial populations together with determining any impact on the sensory properties of chicken meat. The optimal concentration and pH of the ASC solution was found to be 0.8 g ASC/L at pH 2.5, which was suitable for use for the commercial sanitization of chicken meat during processing.

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Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica

Cited by 231 publications

References 25 publications

Outbreaks of antimicrobial resistantSalmonellaCholeraesuis in wild boars piglets from central‐western Spain

Outbreaks of antimicrobial resistantSalmonellaCholeraesuis in wild boars piglets from central‐western Spain

Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

Optimization of an Acidified Sodium Chlorite Solution for Reducing Pathogenic Bacteria and Maintaining Sensory Characteristics of Poultry Meat in Simulation Slaughter Process

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