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

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.

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Short Communication: Detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products using Real-Time PCR Multiplex Method

Asian Journal of Natural Product Biochemistry ◽

10.13057/biofar/f190103 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Alfi Sophian ◽

RATNA PURWANINGSIH ◽

EKA PUTRI JUNIARTI IGIRISA ◽

MUHAMMAD LUTHFI AMIRULLAH ◽

BERTHA LOLO LUKITA ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Salmonella Typhimurium ◽

Real Time ◽

Melting Temperature ◽

Real Time Pcr ◽

Meat Products ◽

Processed Meat ◽

Ct Value ◽

Pcr Multiplex ◽

Pcr Method

Abstract. Sophian A, Purwaningsih R, Igirisa RPJ, Amirullah ML, Lukita BL, Fitri RA. 2020. Short Communication: Detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products using Real-Time PCR Multiplex Method. Asian J Nad Prod Biochem 21: 17-20. The detection of Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 in processed meat products was carried out using Multiplex Real-Time PCR (qPCR) in the Microbiology and Molecular Biology Laboratory at the Indonesian Food and Drug Authority in Gorontalo. The purpose of this study was to provide alternative testing methods for food products circulating in the market. The sample consisted of 25 samples of processed meat products spike with Salmonella typhimurium ATCC 14028 phase 2 and Listeria monocytogenes ATCC 7644 phase 2. The method used in the study was qPCR analysis using the SYBR Green method, while DNA isolation used the direct PCR method. Data analysis was carried out based on Cycle threshold and Melting temperature based on two main criteria. Cycle threshold (Ct) analysis determines the Ct value of the sample and comparing it with the control. Melting temperature (Tm) analysis determines the temperature at which 50% of double-stranded DNA changed to a single standard and comparing it with the melting temperature of positive control. The results showed Salmonella typhimurium ATCC 14028 in the processed meat was detected at an average Ct value of 10.34, and a Tm value of 85.70. The presence of Listeria monocytogenes ATCC 7644 in the samples was recognized at an average Ct value of 14.04, and an average Tm value of 80.07. It can be concluded that the real-time multiplex PCR method can be used to detect Salmonella typhimurium ATCC 14028 and Listeria monocytogenes ATCC 7644 by using the melting curve (Tm) analysis.

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ISOLATION OF LISTERIA MONOCYTOGENES DNA FROM MEAT PRODUCTS FOR QUANTITATIVE DETECTION BY REAL-TIME PCR

Journal of Rapid Methods & Automation in Microbiology ◽

10.1111/j.1745-4581.2006.00058.x ◽

2006 ◽

Vol 14 (4) ◽

pp. 395-404 ◽

Cited By ~ 12

Author(s):

DAVID RODRÍGUEZ-LÁZARO ◽

MARTA HERNÁNDEZ

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Meat Products ◽

Quantitative Detection

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A Fast and Reliable Real-Time PCR Method for Detection of Ten Animal Species in Meat Products

Journal of Food Science ◽

10.1111/1750-3841.14001 ◽

2018 ◽

Vol 83 (2) ◽

pp. 258-265 ◽

Cited By ~ 6

Author(s):

Lissandra Sousa Dalsecco ◽

Rafael Melo Palhares ◽

Pollyana Carvalho Oliveira ◽

Lilian Viana Teixeira ◽

Marcela Gonçalves Drummond ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Animal Species ◽

Meat Products ◽

Pcr Method

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Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products

Frontiers in Microbiology ◽

10.3389/fmicb.2021.668824 ◽

2021 ◽

Vol 12 ◽

Author(s):

Antonia Kreitlow ◽

André Becker ◽

Marwa F. E. Ahmed ◽

Sophie Kittler ◽

Ulrich Schotte ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Meat Products ◽

Lamp Assay ◽

Culture Method ◽

Campylobacter Coli ◽

Standard Culture ◽

One Step ◽

Meat Samples ◽

Minced Meat

A loop-mediated isothermal amplification (LAMP) assay system was established, allowing rplD gene-based simultaneous detection of Campylobacter jejuni and Campylobacter coli in enriched meat products. Additionally, one-step differentiation of target species on agar plates was enabled by cdtC gene- and gyrA gene-based duplex LAMP. Both the rplD and cdtC–gyrA LAMP assays amplified the target sequences in all 62 C. jejuni and 27 C. coli strains used for determining inclusivity and revealed 100% exclusivity toward 85 tested non-target species. Throughout the entire experiments, C. jejuni and C. coli strains were 100% distinguishable by melting curves of cdtC and gyrA LAMP products. After 24-h enrichment, the rplD LAMP assay reliably detected initial inoculation levels of 10–100 CFU/g in artificially contaminated minced meat. Investigation of naturally contaminated meat samples revealed a diagnostic accuracy of 95% toward real-time PCR and 94.1% toward the standard culture method applying the 24-h incubation period. Diagnostic sensitivity and specificity, and positive and negative predictive values were 89.8, 100, 100, and 91.2%, respectively, when measured against real-time PCR, and 89.6, 98.1, 97.7, and 91.2%, respectively, when measured against the standard culture method. After 48-h enrichment, the detection limit of the rplD LAMP assay improved to initial inoculation levels of 1–10 CFU/g in artificially contaminated minced meat. Applying the 48-h incubation period on naturally contaminated meat samples resulted in 100% concordant results between rplD LAMP, real-time PCR, and the standard culture method. The established LAMP assay system was proved to be suitable for rapid meat sample screening. Furthermore, it constitutes a promising tool for investigating other Campylobacter sources and could therefore make a valuable contribution to protect consumers from foodborne illness.

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Comparative Studies of a Real-Time PCR Method and Three Enzyme-Linked Immunosorbent Assays for the Detection of Central Nervous System Tissues in Meat Products†

Journal of Food Protection ◽

10.4315/0362-028x-71.10.2059 ◽

2008 ◽

Vol 71 (10) ◽

pp. 2059-2066 ◽

Cited By ~ 3

Author(s):

HOLGER SCHÖNENBRÜCHER ◽

KATRIN ANNETTE GÖBEL ◽

AMIR ABDULMAWJOOD ◽

JÜRGEN A. RICHT ◽

MICHAEL BÜLTE

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Real Time ◽

Real Time Pcr ◽

Meat Products ◽

Spongiform Encephalopathy ◽

Pcr Assay ◽

Heat Treated ◽

Pcr Method ◽

Minced Meat

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.

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

Journal of Food Protection ◽

10.4315/0362-028x-69.12.2896 ◽

2006 ◽

Vol 69 (12) ◽

pp. 2896-2901 ◽

Cited By ~ 27

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.

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Methods of Detecting Meat Species in Food of Animal Origin

Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Food Science and Technology ◽

10.15835/buasvmcn-fst:2018.0009 ◽

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00405-08 ◽

2008 ◽

Vol 74 (19) ◽

pp. 6060-6067 ◽

Cited By ~ 82

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.

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Real-time PCR High-resolution Melting Analysis for the Species Identification of Meat Products: Focusing on Food Safety and Detection of Meat Adulterations

Thrita ◽

10.5812/thrita.112550 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Peyman Gholamnezhad ◽

Hamed Ahari ◽

Gholamreza Nikbakht Brujeni ◽

Seyed Amir Ali Anvar ◽

Abbas Ali Motalebi

Keyword(s):

High Resolution ◽

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

High Resolution Melting ◽

Meat Products ◽

Hrm Analysis ◽

Melting Analysis ◽

Meat Species ◽

Meat Samples

Background: Real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis are currently considered as reliable techniques for the species identification of meat-based products and widely used to detect meat adulteration. Objectives: To examine the validity of real-time PCR and HRM analysis to identify meat species in meat-based products. Methods: Meat samples from five species (i.e., cattle, sheep, chicken, turkey, and wild pig) were purchased. Minced meat from the animal species of interest was prepared at the purities of 10%, and 20% and also were prepared as single and mixtures of two species. For molecular assessments, DNA samples were extracted from all the meat samples and subjected to real-time PCR by amplifying a mitochondrial cytochrome b specific for each species. Results: All the meat species studied in this research were successfully detected in the mixed meat samples when separately examined by real-time PCR. High-resolution melting analysis showed that all the meat species of interest were efficiently distinguished when examined simultaneously. Conclusions: The data presented here shows that the real-time PCR and HRM analysis are reliable methods for the identification of meat species used in meat products.

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