Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

2004 ◽  
Vol 233 (2) ◽  
pp. 257-267 ◽  
Author(s):  
D RODRIGUEZLAZARO
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection

scholarly journals Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly, iap, and lin02483 Targets and AmpliFluor Technology

2004 ◽  
Vol 70 (3) ◽  
pp. 1366-1377 ◽  
Author(s):  
David Rodr�guez-L�zaro ◽  
Marta Hern�ndez ◽  
Mariela Scortti ◽  
Teresa Esteve ◽  
Jos� A. V�zquez-Boland ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Genes ◽  
Dynamic Range ◽  
Significant Proportion ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Food Borne ◽  
Highly Sensitive

ABSTRACT We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R 2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

scholarly journals Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

2005 ◽  
Vol 71 (4) ◽  
pp. 2190-2194 ◽  
Author(s):  
Morgan Guilbaud ◽  
Pierre de Coppet ◽  
Fabrice Bourion ◽  
Cinta Rachman ◽  
Hervé Prévost ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Method ◽  
Sybr Green ◽  
Sybr Green I ◽  
Quantitative Detection ◽  
Pcr Assay

ABSTRACT A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2.

scholarly journals Rapid Quantitative Detection of Listeria monocytogenes in Meat Products by Real-Time PCR

2004 ◽  
Vol 70 (10) ◽  
pp. 6299-6301 ◽  
Author(s):  
David Rodríguez-Lázaro ◽  
Anna Jofré ◽  
Teresa Aymerich ◽  
Marta Hugas ◽  
Maria Pla
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Meat Products ◽  
Plate Count ◽  
Quantitative Detection ◽  
Simple Method ◽  
Promising Alternative ◽  
Chelex 100 ◽  
Plate Count Method

ABSTRACT We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.

Rapid Quantitative Detection of Listeria monocytogenes in Salmon Products: Evaluation of Pre–Real-Time PCR Strategies

2005 ◽  
Vol 68 (7) ◽  
pp. 1467-1471 ◽  
Author(s):  
DAVID RODRÍGUEZ-LÁZARO ◽  
ANNA JOFRÉ ◽  
TERESA AYMERICH ◽  
MARGARITA GARRIGA ◽  
MARIA PLA
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Measures ◽  
Plate Count ◽  
Quantitative Detection ◽  
Promising Alternative ◽  
Fish Products ◽  
Smoked Fish ◽  
Seafood Processing

The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

2009 ◽  
Vol 26 (3) ◽  
pp. 311-316 ◽  
Author(s):  
Oscar Fernando D'Urso ◽  
Palmiro Poltronieri ◽  
Santo Marsigliante ◽  
Carlo Storelli ◽  
Marta Hernández ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Food Samples ◽  
Quantitative Detection ◽  
Pcr Method

scholarly journals Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

2000 ◽  
Vol 66 (10) ◽  
pp. 4266-4271 ◽  
Author(s):  
Hege Karin Nogva ◽  
Knut Rudi ◽  
Kristine Naterstad ◽  
Askild Holck ◽  
Dag Lillehaug
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Skim Milk ◽  
Raw Milk ◽  
Magnetic Bead ◽  
Quantitative Detection ◽  
Listeriolysin O ◽  
Pure Cultures

ABSTRACT PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenesbased on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all otherListeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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