scholarly journals Real-Time PCR Method for Detection of Pathogenic Yersinia enterocolitica in Food

2008 ◽  
Vol 74 (19) ◽  
pp. 6060-6067 ◽  
Author(s):  
S. Thisted Lambertz ◽  
C. Nilsson ◽  
S. Hallanvuo ◽  
M. Lindblad
Keyword(s):  
Real Time ◽  
Yersinia Enterocolitica ◽  
Real Time Pcr ◽  
False Negative ◽  
Pcr Amplification ◽  
Reference Method ◽  
Detection Methods ◽  
Good Precision ◽  
Diverse Range ◽  
Pcr Method

ABSTRACT The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.

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

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Kuppuswamy N. Kasturi ◽  
Tomas Drgon
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
False Negative ◽  
Screening Method ◽  
Reference Method ◽  
Rapid Screening ◽  
Content Type ◽  
Pcr Method ◽  
Group D

ABSTRACT 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 non-Enteritidis 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.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

Rapid real-time PCR assay for detecting Salmonella in raw and ready-to-eat meats

2008 ◽  
Vol 56 (4) ◽  
pp. 451-458 ◽  
Author(s):  
Jitu Patel ◽  
Arvind Bhagwat
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Molecular Beacon ◽  
False Negative ◽  
Detection Methods ◽  
Time Data ◽  
Pcr Assay ◽  
Serovar Typhimurium ◽  
Meat Samples

A real-time PCR assay was evaluated for the rapid detection (10 h) ofSalmonellain meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated withSalmonella entericaserovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

scholarly journals SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A

2007 ◽  
Vol 73 (9) ◽  
pp. 2891-2896 ◽  
Author(s):  
Lucia Fenicia ◽  
Fabrizio Anniballi ◽  
Dario De Medici ◽  
Elisabetta Delibato ◽  
Paolo Aureli
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clostridium Botulinum ◽  
False Negative ◽  
Type A ◽  
Sybr Green ◽  
Mouse Bioassay ◽  
Chelex 100 ◽  
Microbiological Methods ◽  
Pcr Method

ABSTRACT Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

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.

Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods

2017 ◽  
Vol 20 (3) ◽  
pp. 477-484 ◽  
Author(s):  
M.A. Stachelska
Keyword(s):  
Real Time ◽  
Yersinia Enterocolitica ◽  
Real Time Pcr ◽  
Culture Method ◽  
Molecular Techniques ◽  
Pork Meat ◽  
Culture Methods ◽  
The Real ◽  
Pcr Method ◽  
Classical Culture

AbstractThe aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5’ nuclease PCR protocol. The probe was labelled at the 5’ end with the fluorescent reporter dye (FAM) and at the 3’ end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

scholarly journals Degradation of 4-Chloro-2-Methylphenoxyacetic Acid in Top- and Subsoil Is Quantitatively Linked to the Class III tfdA Gene

2006 ◽  
Vol 72 (2) ◽  
pp. 1476-1486 ◽  
Author(s):  
Jacob Bælum ◽  
Trine Henriksen ◽  
Hans Christian Bruun Hansen ◽  
Carsten Suhr Jacobsen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Dry Weight ◽  
Initial Assessment ◽  
Class Iii ◽  
Gradient Gel Electrophoresis ◽  
Pcr Method ◽  
Or Genes

ABSTRACT The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g−1 soil to approximately 3 × 104 genes g−1 soil and to 7 × 105 genes g−1 soil for topsoil initially added to 2.3 mg MCPA kg−1 (dry weight) soil and 20 mg MCPA kg−1 (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.

scholarly journals Detection of food hazards in foods: comparison of real time polymerase chain reaction and cultural methods

2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Paolo Bonilauri ◽  
Lia Bardasi ◽  
Roberto Leonelli ◽  
Mattia Ramini ◽  
Andrea Luppi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Food Samples ◽  
Alternative Methods ◽  
Animal Origin ◽  
Screening Methods ◽  
Molecular Screening ◽  
Cultural Methods ◽  
Pcr Method

Foodstuffs should not contain microorganisms or their toxins or metabolites in quantities suggesting an unacceptable risk for human health. The detection of food hazards in foods is performed by several tests that produce results dependent on the analytical method used: an analytical reference method, defined as standard, is associated with each microbiological criterion laid down in Regulation 2073/2005, but, analytical methods other than the reference ones, in particular more rapid methods, could be used. Combined screening methods performed by real time PCR are currently validated as alternative methods according to the ISO 16140:2003 and certified by the Association Française de Normalisation. However, the positive results obtained with these alternative methods, the investigated molecular relations that resulted positive have to be confirmed with cultural methods using the same enrichment media in which the molecular screening was performed. Since it is necessary to assess if these testing schemes provide equivalent guarantees of food safety, the aim of this retrospective study is to analyze the data collected, from 2012 to 2014, by Emilia Romagna Region in the field of <em>Piano Regionale Alimenti</em> (Food Regional Plan), during official controls monitoring food samples, of animal and other than animal origin. Records performed by combined methods of molecular screening of <em>Salmonella</em> spp., <em>Listeria monocytogenes</em> and thermophilic <em>Campylobacter</em> and cultural confirmation results were gathered together and the results were compared in order to assess the sensitivity of the methods. A total of 10.604 food samples were considered in this study: the comparison of the data revealed that the RT-PCR method detected <em>Salmonella</em>, <em>L. monocytogenes</em>, and thermophilic <em>Campylobacter</em> in 2.18, 3.85 and 3.73% of the samples, respectively, whereas by using cultural method these pathogens were isolated in 0.43, 1.57 and 1.57 % of samples, respectively. In spite of the use of the same enrichment broth, the real time PCR method disclosed a percentage of positive samples that were negative to cultural examination ranging between 20 and 43%, with a PCR/culture ratio between 2.37 to 5.00. In conclusion, the results of this study pose a doubt about the sensitivity of the official cultural methods regarding the isolation of the three investigated foodborne pathogens. Moreover this study may be a useful tool for Veterinary Authorities to assess appropriate sampling plans to control the risk relating to the consumption of contaminated foods.

scholarly journals Real-Time PCR Method Combined with a Matrix Lysis Procedure for the Quantification of Listeria monocytogenes in Meat Products

Foods ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 735
Author(s):  
Mirian Labrador ◽  
Carlota Giménez-Rota ◽  
Carmen Rota
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Foodborne Pathogen ◽  
Meat Products ◽  
Reference Method ◽  
Meat Industry ◽  
Gene Copies ◽  
Pcr Method ◽  
Meat Samples

In this study a real-time PCR method has been developed for the specific quantification of the foodborne pathogen Listeria monocytogenes on meat products through the gene hlyA. The PCR was combined with a matrix lysis that allowed the obtaining of the microorganisms without sample dilution and the elimination the PCR inhibitors from dry-cured ham. The qPCR method calibration curve had an efficiency of 100.4%, limits of detection and quantification were 30.1 ± 6.2 CFU/g which is under the legal limit of L. monocytogenes in ready-to-eat products, and an analytical variability <0.25 log hlyA gene copies/reaction. The analysis was performed simultaneously with the reference method ISO 11290-2. The comparison of the qPCR-matrix lysis results with the reference method showed an excellent correspondence, with a relative accuracy between 95.83–105.20%. Finally, the method was applied to commercial derived meat samples and the pathogen was quantified in one of the commercial samples assayed in 69.1 ± 13.9 CFU/g while the reference method did not quantify it. The optimized qPCR showed higher precision and sensitivity than the reference method at low concentrations of the microorganism in a shorter time. Therefore, qPCR-matrix lysis shows a potential application in the meat industry for L. monocytogenes routine control.

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.

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