Rapid Detection of Listeria monocytogenes by Real-Time PCR in Processed Meat and Dairy Products

2014 ◽  
Vol 77 (3) ◽  
pp. 453-458 ◽  
Author(s):  
EUN JEONG HEO ◽  
BO RA SONG ◽  
HYUN JUNG PARK ◽  
YOUNG JO KIM ◽  
JIN SAN MOON ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Processed Meat ◽  
Ground Meat ◽  
Rt Pcr ◽  
Hybridization Probe ◽  
Cell Counts ◽  
Two Samples

The objectives of this study were to evaluate the detection of Listeria monocytogenes in different ready-to-eat foods using real-time PCR (RT-PCR). Various concentrations (100 to 105 CFU/ml) of L. monocytogenes ATCC 19115 were inoculated into ham, sausage, ground meat, processed milk, cheese, and infant formula. L. monocytogenes ATCC 19115 in the samples was then enumerated on Oxford agar, and DNA was extracted from the samples before and after incubation at 36°C for 4 h. A set of primers and hybridization probe designed in this study was then used to detect the pathogen. The standard curve was then prepared by plotting cycle threshold values for each dilution versus L. monocytogenes cell counts (log CFU). The specificity of the set of primers and hybridization probe was appropriate. A 4-h incubation at 36°C before DNA extraction produced optimum standard curves in comparison to the results for a 0-h incubation. Thus, a 4-h incubation at 36°C was applied for monitoring L. monocytogenes in collected food samples. To monitor L. monocytogenes in foods, 533 samples (ham, 129; sausage, 226; ground meat, 72; processed cheese, 54; processed milk, 42; and infant formula, 10) were collected from retail markets and from the step before pasteurization in plants. Of all 533 samples, 4 samples (0.8%) showed positive signals in RT-PCR. Two samples from hams (1.6%) and two samples from sausages (0.9%) were determined to be positive for L. monocytogenes at <100 CFU/g. The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes in processed meat and milk products.

scholarly journals Prevalence of Bovine viral diarrhea virus in cattle herds from Basrah and Nassirya Provinces by direct and indirect Elisa and Real time qPCR

2012 ◽  
Vol 11 (2) ◽  
pp. 1
Author(s):  
B. A. Jarullah, J. Aed Gati, and A. Saleh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Indirect Elisa ◽  
Positive Sample ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Virus Rna ◽  
Two Samples ◽  
Cattle Herds

The current study was conducted to investigate the prevalence of BVD virus in Basrah and Nassirya city by using ELISA and RT-PCR. Two hundreds and eighty two samples of non vaccinated cattle sera samples collected from two regions of Iraq (188 samples from Nassirya city and 92 samples from Basrah city). Samples tested by Enzyme Linked Immunosorbent Assay (ELISA) antigen capture. Positive results were 20 samples ( 8 sample in Thi-Qar and 12 positive samples from Basrah). All samples submitted to indirect ELISA(IDEXX HerdCheck ELISA )for detect BVDV antibodies .Genotyping of all 20 positive samples to antigen detection were tested by Real time PCR, using Cador BVDV ½ kit, after extraction of virus RNA by QIAamp mini kit. The results revealed that there were 20 positive sample according to direct ELISA(Ag detection), while 66 sample were positive to indirect ELISA, as well as, the result of RT-PCR showed that there were two sample positive to BVDV type-1 (one sample form each city).Key words: BVDV, Genotype, ELISA, Iraq, Real time PCR.

scholarly journals Συγκριτική μελέτη των ISO καλλιεργητικών μεθόδων και των μοριακών μεθόδων PCR και RT-PCR στην ανίχνευση Listeria monocytogenes από τρόφιμα

2007 ◽  
Author(s):  
Αικατερίνη Σαλαμούρα
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Rt Pcr

Η L. monocytogenes έχει απομονωθεί από κλινικά και περιβαλλοντικά δείγματα, καθώς και από δείγματα τροφίμων. Μελέτες που έχουν γίνει σε μεγάλες επιδημίες έχουν δείξει ότι η λιστερίωση είναι τροφιμογενές νόσημα. Η L. monocytogenes έχει απομονωθεί από ποικιλία τροφίμων, καθώς και από τις γραμμές παραγωγής κρύας ή θερμής κάπνισης ιχθύων, γι αυτό το λόγο τα ιχθυηρά είναιυπεύθυνα για περιπτώσεις ανθρώπινης λιστερίωσης. Οι αριθμοί των κυττάρων της L. monocytogenes από τα τρόφιμα, τα περιβαλλοντικά και κλινικά δείγματα είναι πολύ μικροί, γι αυτό είναι πολύ σημαντική η επιλογή της κατάλληλης μεθόδου για την ανίχνευση του βακτηρίου. Σκοπός της παρούσας μελέτης ήταν: α) η συγκριτική μελέτη ανίχνευσης της L. monocytogenes από τα τρόφιμα με τη χρήση της ISO 11290-1 καλλιεργητικής μεθόδου και των νέων μοριακών μεθόδων (PCR, Real Time PCR) και β) ο προσδιορισμός της συχνότητας απομόνωσης της L. monocytogenes σε ιχθυηρά της ελληνικής αγοράς, τόσο με τη χρήση κλασικών καλλιεργητικών υποστρωμάτων, όσο και νέων μοριακών τεχνικών. Για τη συγκριτική μελέτη, χρησιμοποιήθηκαν υγρά υποστρώματα, στα οποία ενοφθαλίστηκε πρότυπο στέλεχος L. monocytogenes γνωστής συγκέντρωσης. Για τηνανίχνευση του μικροοργανισμού χρησιμοποιήθηκε η ISO 11290-1 καλλιεργητική μέθοδος (σε Oxford άγαρ και σε PALCAM άγαρ) και η Αλυσιδωτή Αντίδραση Πολυμεράσης (PCR), με χρήση δύο νέων αυτοματοποιημένων συστημάτων, του LightCycler (Roche) και του Bax® system (Qualicon). Εξετάστηκαν διαδοχικές αραιώσεις του αρχικού εναιωρήματος της L. monocytogenes και το πείραμαεπαναλήφθηκε με τα δείγματα να έχουν υποστεί νωρίτερα παστερίωση και αποστείρωση. Όλα τα πειράματα πραγματοποιήθηκαν εις διπλούν. Για τον προσδιορισμό της συχνότητας της L. monocytogenes στα ιχθυηρά, εξετάστηκαν 255 δείγματα νωπών και επεξεργασμένων ιχθύων, γλυκών και αλμυρών υδάτων, τόσο με τη χρήση των κλασικών καλλιεργητικών υποστρωμάτων (ISO11290-1), όσο και των δύο νέων μοριακών τεχνικών. Τα δείγματα συλλέχθηκαν από σούπερ μάρκετ, ιχθυοπωλεία και ιχθυοτροφεία της Ηπείρου. To Half-Fraser broth και το Fraser broth χρησιμοποιήθηκαν ως προ-εμπλουτιστικό και εμπλουτιστικό θρεπτικό μέσο αντίστοιχα, ενώ το Oxford άγαρ και το PALCAM άγαρ (ISO 11290-1) ως εκλεκτικά στερεά θρεπτικά υποστρώματα. Με τα καλλιεργητικά μέσα απομονώθηκαν επτά στελέχη L. monocytogenes (2,75%), τα οποία επίσης ανιχνεύθηκαν και από τα δύο μοριακά συστήματα PCR. Το ένα στέλεχος L. monocytogenes (ορότυπος 4) απομονώθηκε από ένα φρέσκο κέφαλο (3,3%), δύο στελέχη L. monocytogenes(ορότυπος 1) απομονώθηκαν από εισαγόμενα φιλέτα σολομού (Νορβηγίας) (6,7%) και τέσσερα στελέχη L. monocytogenes (ορότυπος 1) απομονώθηκαν από καπνιστές πέστροφες (8,9%). Όλα τα στελέχη ήταν ευαίσθητα στα αντιβιοτικά πενικιλίνη και αμπικιλίνη. Από τα αποτελέσματα φαίνεται ότι οι μοριακές τεχνικές είναι πιο γρήγορες και ακριβείς σε σχέση με την καλλιεργητική μέθοδο. Η ανίχνευση και ταυτοποίηση της L. monocytogenes μπορεί να γίνει σε δύο ημέρες σε σύγκριση με τιςκαλλιεργητικές τεχνικές που απαιτούν περίπου μία εβδομάδα. Από τα αποτέλεσμα φαίνεται επίσης ότι τα ιχθυηρά μπορούν να αποτελέσουν μέσο μετάδοσης της L. monocytogenes στον άνθρωπο, είτε μέσω κατανάλωσης μη κάλο μαγειρεμένων ιχθύων, είτε με επιμόλυνση από μαγειρικά σκεύη.

scholarly journals Enumeration of Viable Listeria monocytogenes Cells by Real-Time PCR with Propidium Monoazide and Ethidium Monoazide in the Presence of Dead Cells

2007 ◽  
Vol 73 (24) ◽  
pp. 8028-8031 ◽  
Author(s):  
Y. Pan ◽  
F. Breidt
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Antimicrobial Effect ◽  
Viable Cell ◽  
Propidium Monoazide ◽  
Ethidium Monoazide ◽  
Cell Counts

ABSTRACT Propidium monoazide (PMA) and ethidium monoazide were used for enumeration of viable Listeria monocytogenes cells in the presence of dead cells. PMA had no antimicrobial effect on L. monocytogenes. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells from planktonic and biofilm sources over a 4-log range.

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

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.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

scholarly journals Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

2012 ◽  
Vol 97 (9) ◽  
pp. 4021-4037 ◽  
Author(s):  
Elodie Barbau-Piednoir ◽  
Nadine Botteldoorn ◽  
Marc Yde ◽  
Jacques Mahillon ◽  
Nancy H. Roosens
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Development And Validation

scholarly journals A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1702
Author(s):  
Arkadiusz Dors ◽  
Ewelina Czyżewska-Dors ◽  
Grzegorz Woźniakowski
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Finishing Pigs ◽  
Lawsonia Intracellularis ◽  
Fecal Samples ◽  
Brachyspira Pilosicoli ◽  
Brachyspira Hyodysenteriae ◽  
Veterinary Research ◽  
Two Samples ◽  
Intestinal Pathogens

Background: The major pathogenic intestinal spirochetes affecting pigs during the growing- finishing stage of production include Brachyspira hyodysenteriae and Brachyspira pilosicoli. Infections by these pathogens, which affect the economics of pig production, can result in mortality, growth rate losses and substantial antibiotic costs. The aim of this study was to assess the current occurrence of B. hyodysenteriae and B. pilosicoli in Polish pig herds. Moreover, associations between the presence of diarrhea or other intestinal pathogens and occurrence of B. hyodysenteriae and B. pilosicoli in pigs were investigated. Methods: Between January 2017 and August 2019, a total of 401 samples of pig feces from 95 different herds were submitted to the National Veterinary Research Institute of Poland. These samples were obtained from pigs older than 7 weeks. All the received fecal samples were examined for the presence of B. hyodysenteriae, B. pilosicoli and Lawsonia intracellularis by real-time PCR. Results: For B. pilosicoli, 4.5% (95% CI, 2.5–7.0%) of samples and 13.7% (95% CI, 7.5–22.3%) of herds were positive. Out of 12 samples, B. pilosicoli was detected simultaneously with L. intracellularis, B. hyodysenteriae and B. pilosicoli were detected alone in two samples each. In terms of B. hyodysenteriae, 7.0% of samples (95% CI, 4.7–9.9%) from 18.9% of herds (95% CI, 11.6–28.3%) were positive in real time PCR. The presence of B. hyodysenteriae in fecal samples was associated with the presence of diarrhea in pigs. Conclusions: This study confirmed that B. pilosicoli infections occur in Polish pig herds, but the prevalence is at a low level and the presence of B. pilosicoli is not associated with the development of diarrhea in pigs. B. hyodysenteriae is still a common cause of diarrhea among pigs from Polish herds.

scholarly journals Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

2007 ◽  
Vol 29 (4) ◽  
pp. 289-299
Author(s):  
Robert E. Page ◽  
Andrés J. P. Klein-Szanto ◽  
Samuel Litwin ◽  
Emmanuelle Nicolas ◽  
Raid Al-Jumaily ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
Real Time ◽  
Cell Lines ◽  
Cancer Cell ◽  
Real Time Pcr ◽  
Ovarian Tumor ◽  
Cancer Cell Lines ◽  
Rt Pcr ◽  
Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

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