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.

Detection of Salmonella by Flow-Through Immunocapture Real-Time PCR in Selected Foods within 8 Hours

2007 ◽  
Vol 70 (4) ◽  
pp. 1002-1006 ◽  
Author(s):  
BENJAMIN R. WARREN ◽  
HYUN-GYUN YUK ◽  
KEITH R. SCHNEIDER
Keyword(s):  
Real Time ◽  
Drug Administration ◽  
Real Time Pcr ◽  
Food And Drug Administration ◽  
Ground Beef ◽  
Culture Method ◽  
Food Samples ◽  
Flow Through ◽  
Pcr Method

This study investigated flow-through immunocapture (FTI), using the Pathatrix device, followed by plating on xylose lysine desoxycholate (XLD) agar (FTI-XLD) or analysis by real-time PCR (FTI-PCR) for the detection of Salmonella on smooth tomato surfaces and in potato salad and ground beef within 8 h. Food samples were inoculated with an appropriate dilution of a five-serovar Salmonella cocktail and enriched for 5 h. Following enrichment, samples were analyzed by the FTIXLD and FTI-PCR methods. Food samples were also analyzed by a modified U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Salmonella culture method for comparison. Salmonella inoculated at 100 CFU per tomato or 100 CFU/25 g was detected by the FTI-XLD method in 6, 8, and 4 of 10 samples for tomatoes, potato salad, and ground beef, respectively. Salmonella inoculated at 100 CFU per tomato or 100 CFU/25 g was detected by the FTI-PCR method in 8, 9, and 9 of 10 samples for tomatoes, potato salad, and ground beef, respectively. The FTI-PCR method achieved significantly higher (P &lt; 0.05) detection of Salmonella on tomatoes, whereas the FTI-XLD method achieved significantly lower (P &lt; 0.05) detection of Salmonella in ground beef when compared with the modified BAM Salmonella culture method; however, all other comparisons to the modified BAM method were not significantly different. The FTI-XLD method demonstrated the ability to isolate presumptive Salmonella colonies up to 48 h faster than did the modified BAM Salmonella culture method.

scholarly journals Methods of Detecting Meat Species in Food of Animal Origin

2018 ◽  
Vol 75 (2) ◽  
pp. 125
Author(s):  
Lavinia-Maria CHIŞ ◽  
Dan-Cristian VODNAR
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat Product ◽  
Meat Products ◽  
Ease Of Use ◽  
Animal Origin ◽  
Processed Meat ◽  
Elisa Method ◽  
Food Of Animal Origin ◽  
Pcr Method

An important factor in the detection of falsification is the control of the composition of the meat at each stage of manufacturing the product. The PCR method is based on the study of proteins and meat nucleic acids used in food for the detection of animal species. Another technique is the Elisa method that works on the principle of identification and measurement of the quantity of molecules in a sample. There are several types of Elisa to increase specificity due to differences in structure and sample characteristics. By comparing the two methods used to identify the processed meat product species, Real Time PCR had the highest prediction as results. However, the Elisa method is more time efficient and easier to use. Real Time PCR is effective in identifying processed meat products that require low detection. The Elisa Kit is useful because of the ease of use.

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 Application of real-time PCR for DNA identification of ruminants in raw animal products

2018 ◽  
Vol 74 (1) ◽  
pp. 6061-2018
Author(s):  
ANNA WEINER ◽  
ILONA PAPROCKA ◽  
AGATA GOŁĘBIOWSKA ◽  
KRZYSZTOF KWIATEK
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Animal Origin ◽  
Dna Identification ◽  
Animal Products ◽  
Modified Method ◽  
Pcr Method ◽  
Processed Products ◽  
By Products

According with current regulations it is possible export of processed animal proteins produced from tissues other animals than ruminants. In order to ensure appropriate control studies were undertaken to adopt of method real-time PCR used for analysis of processed products of animal origin. The material consisted of raw animal by-products of beef, pork and poultry. In the assays mitochondrial DNA was used. Modified method allows to detect the DNA of ruminants at the level of 0.025%. Based on obtained results of the validation studies were found that the real-time PCR method can be used in routine tests for identification of ruminant DNA in raw animal by-products..

scholarly journals Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit in Combination with ShortPrep foodproof II Kit: Performance-Tested MethodSM 070401

2006 ◽  
Vol 89 (2) ◽  
pp. 374-398 ◽  
Author(s):  
Benjamin Junge ◽  
Kornelia Berghof-Jger ◽  
Joseph A Odumeru ◽  
Denise A Kaji
Keyword(s):  
Real Time ◽  
False Positive ◽  
Stress Test ◽  
Reference Method ◽  
Food Samples ◽  
Research Association ◽  
Long Term Study ◽  
Microbiological Methods ◽  
Sensitivity Rate ◽  
Pcr Method

Abstract A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results.For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (110 colony-forming units (CFU)/25 g) and 20 samples with a high level (1050 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook.After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCRmethod resulted inmore positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4; for high inoculated foodstuffs: 7.1). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes.The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes.In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI.The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100.Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results.The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lotswere tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results.The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAMmethods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAMculture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.

Rapid Detection of Salmonella in Foods Using Real-Time PCR

2008 ◽  
Vol 71 (12) ◽  
pp. 2436-2441 ◽  
Author(s):  
CHORNG-MING CHENG ◽  
WEN LIN ◽  
KHANH THIEN VAN ◽  
LIEUCHI PHAN ◽  
NELLY N. TRAN ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Samples ◽  
Taqman Probe ◽  
Cell Culturing ◽  
Chili Powder ◽  
Pcr Method ◽  
Food Matrices ◽  
Salmonella Serovars

Conventional methods for detection of Salmonella serovars in foods are generally time-consuming and labor intensive. A real-time PCR method has been developed with custom designed primers and a TaqMan probe to detect the presence of a 262-bp fragment of the Salmonella-specific invA gene. The method has been tested with a total of 384 field-isolated Salmonella serovars and non-Salmonella stock strains, as well as 420 U.S. Food and Drug Administration food samples, comprising a variety of food matrices. The method was highly specific in detecting Salmonella in spiked chili powder and shrimp samples, with a sensitivity of 0.04 CFU/g. In addition, the method is faster, more accurate, and less costly than the traditional U.S. Food and Drug Administration's Bacteriological Analytical Manual cell-culturing and the AOAC International–approved VIDAS methods to detect Salmonella in foods.

scholarly journals Diagnostic Real-Time PCR for Detection of Salmonella in Food

2004 ◽  
Vol 70 (12) ◽  
pp. 7046-7052 ◽  
Author(s):  
Burkhard Malorny ◽  
Elisa Paccassoni ◽  
Patrick Fach ◽  
Cornelia Bunge ◽  
Annett Martin ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Traditional Culture ◽  
Culture Method ◽  
Raw Milk ◽  
Food Samples ◽  
Diagnostic Methods ◽  
Analysis Time ◽  
Diagnostic Pcr ◽  
Pcr Method

ABSTRACT A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 103 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 104 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document