Assessment of Listeria sp. Interference Using a Molecular Assay To Detect Listeria monocytogenes in Food

2016 ◽  
Vol 79 (1) ◽  
pp. 138-143 ◽  
Author(s):  
SANDRA I. ZITTERMANN ◽  
BRENDA STANGHINI ◽  
RYAN SOO SEE ◽  
ROBERTO G. MELANO ◽  
PETER BOLESZCZUK ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Reference Method ◽  
Food Samples ◽  
Pcr Assay ◽  
The Real ◽  
Current Reference ◽  
Listeria Species

ABSTRACT Detection of Listeria monocytogenes in food is currently based on enrichment methods. When L. monocytogenes is present with other Listeria species in food, the species compete during the enrichment process. Overgrowth competition of the nonpathogenic Listeria species might result in false-negative results obtained with the current reference methods. This potential issue was noted when 50 food samples artificially spiked with L. monocytogenes were tested with a real-time PCR assay and Canada's current reference method, MFHPB-30. Eleven of the samples studied were from foods naturally contaminated with Listeria species other than those used for spiking. The real-time PCR assay detected L. monocytogenes in all 11 of these samples; however, only 6 of these samples were positive by the MFHPB-30 method. To determine whether L. monocytogenes detection can be affected by other species of the same genus due to competition, an L. monocytogenes strain and a Listeria innocua strain with a faster rate of growth in the enrichment broth were artificially coinoculated at different ratios into ground pork meat samples and cultured according to the MFHPB-30 method. L. monocytogenes was detected only by the MFHPB-30 method when L. monocytogenes/L. innocua ratios were 6.0 or higher. In contrast, using the same enrichments, the real-time PCR assay detected L. monocytogenes at ratios as low as 0.6. Taken together, these findings support the hypothesis that L. monocytogenes can be outcompeted by L. innocua during the MFHPB-30 enrichment phase. However, more reliable detection of L. monocytogenes in this situation can be achieved by a PCR-based method mainly because of its sensitivity.

scholarly journals Improvement of Legionnaires’ disease diagnosis using real-time PCR assay: a retrospective analysis, Italy, 2010 to 2015

2018 ◽  
Vol 23 (50) ◽  
Author(s):  
Maria Luisa Ricci ◽  
Antonella Grottola ◽  
Giulia Fregni Serpini ◽  
Antonino Bella ◽  
Maria Cristina Rota ◽  
...  
Keyword(s):  
Real Time ◽  
Legionella Pneumophila ◽  
Real Time Pcr ◽  
Reference Method ◽  
Disease Diagnosis ◽  
Pcr Assay ◽  
Predictive Values ◽  
The Real ◽  
Legionnaires Disease ◽  
Antigen Tests

Aim To evaluate real-time PCR as a diagnostic method for Legionnaires’ disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised. Methods Two independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010–15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated. Results Overall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2–99.6), while the sensitivity was 63.6% (95%CI: 58.6–68.6) and 77.8% (95% CI: 72.9–82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2). Conclusion Regardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case.

Evaluation of an Immunomagnetic Separation–Real-Time PCR Assay for the Rapid Detection of Salmonella in Meat

2006 ◽  
Vol 69 (12) ◽  
pp. 2896-2901 ◽  
Author(s):  
ANGELIKA NOTZON ◽  
REINER HELMUTH ◽  
JOHANN BAUER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Positive Rate ◽  
False Negative ◽  
False Negative Rate ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
Pcr Assay ◽  
Positive Rate ◽  
Meat Samples

The aim of this study was the comparison of an immunomagnetic separation (IMS)–real-time PCR assay for the detection of Salmonella with the cultural reference method according to §35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS–real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe–based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS–real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS–real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance indexκ defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS–real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index κ had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS–real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.

scholarly journals A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study

2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  
Keyword(s):  
Real Time ◽  
Multicenter Study ◽  
Real Time Pcr ◽  
False Negative ◽  
False Negative Result ◽  
Pcr Assay

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Susceptibility of Maize Inbreds and Incidence of Symptomless Infection by the Head Smut Pathogen, Sphacelotheca reiliana

2016 ◽  
Vol 17 (1) ◽  
pp. 1-5 ◽  
Author(s):  
S. J. Anderson ◽  
H. E. Simmons ◽  
R. D. French-Monar ◽  
G. P. Munkvold
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Field Trials ◽  
Growth Stages ◽  
High Susceptibility ◽  
Head Smut ◽  
Pcr Assay ◽  
Disease Symptoms ◽  
The Real ◽  
Seedling Infection

A real-time PCR assay was used to compare seedling infection by Sphacelotheca reiliana, the causal agent of head smut, among five inbred genotypes representing low, moderate, and high susceptibility to the disease. Seeds were coated with teliospores and planted in autoclaved field soil in a growth chamber. Incidence of seedling infection at growth stage V3 differed between an inbred genotype of low susceptibility and those of moderate and high susceptibility, but did not differ between the high and moderately susceptible groups (P < 0.05). The real-time PCR assay was also used to compare infection status at early and late vegetative stages with observable symptoms in the field. We detected infection via real-time PCR in maize at both growth stages during field trials conducted in Texas and California but observed no disease symptoms (smutted ears or tassels). Notably, the fungus was present in up to 31% of the ear shoots in plots without disease symptoms. The real-time assay can be a useful tool for screening seedling-stage host resistance, and for better understanding the progress of infection in different maize genotypes. The field data suggest that asymptomatic infection is much more common than previously thought, and may have important implications for the epidemiology of this fungus under diverse plant resistance and growing conditions. Accepted for publication 11 December 2015. Published 5 January 2016.

A Quantitative Real-time PCR Assay for Quantification of Viable Listeria Monocytogenes Cells After Bacteriocin Injury in Food-First Insights

2010 ◽  
Vol 61 (6) ◽  
pp. 515-519 ◽  
Author(s):  
Antonio Cobo Molinos ◽  
Hikmate Abriouel ◽  
Nabil Ben Omar ◽  
Magdalena Martinez-Canamero ◽  
Antonio Gálvez
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay

Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

2004 ◽  
Vol 67 (11) ◽  
pp. 2424-2429 ◽  
Author(s):  
G. E. KAUFMAN ◽  
G. M. BLACKSTONE ◽  
M. C. L. VICKERY ◽  
A. K. BEJ ◽  
J. BOWERS ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pcr Amplification ◽  
Oyster Tissue ◽  
Pcr Assay ◽  
Colony Hybridization ◽  
The Real ◽  
Mantle Fluid ◽  
June July

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P &lt; 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P &lt; 0.05; and oyster: r = 0.99, P &lt; 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P &lt; 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

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