Detection of Listeria monocytogenes HlyA gene in Food Samples of Meat by Real-Time PCR In Erbil city

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detectListeria monocytogenesin artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. ForListeriaspecies the measuring time was maximum 34 h. The absence ofL. monocytogenescould reliably be proven by the redox potential measurement method, butListeria innocuaandBacillus subtiliscould not be differentiated fromL. monocytogeneson the basis of the redox curves. The presence ofL. monocytogeneshad to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g ofL. monocytogenesin a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection ofL. monocytogenesin food.

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Application of SYBR Green I and TaqMan probe-based real-time PCRs for the identification of Listeria spp. and Listeria monocytogenes

Bulletin of the Veterinary Institute in Pulawy ◽

10.1515/bvip-2015-0073 ◽

2015 ◽

Vol 59 (4) ◽

pp. 489-494 ◽

Cited By ~ 2

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Anna Szczawińska ◽

Monika Reksa ◽

Marek Krupa ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Taqman Probe ◽

Rdna Gene ◽

Intercalating Dye ◽

Hlya Gene

Abstract The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

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A New Rapid Real-Time PCR Method for Detection of Listeria monocytogenes Targeting the hlyA Gene

Food Science and Technology Research ◽

10.3136/fstr.18.47 ◽

2012 ◽

Vol 18 (1) ◽

pp. 47-57 ◽

Cited By ~ 9

Author(s):

Pei LIU ◽

Hiromi MIZUE ◽

Kumiko FUJIHARA ◽

Hiroshi KOBAYASHI ◽

Hideaki KAMIKADO ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Pcr Method ◽

Hlya Gene

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Development of a multiplex real‐time PCR for simultaneous detection of Bacillus cereus , Listeria monocytogenes , and Staphylococcus aureus in food samples

Journal of Food Safety ◽

10.1111/jfs.12558 ◽

2018 ◽

Vol 39 (1) ◽

Cited By ~ 12

Author(s):

Shuai Wei ◽

Eric Banan‐Mwine Daliri ◽

Ramachandran Chelliah ◽

Byung‐Jae Park ◽

Ji‐Su Lim ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Bacillus Cereus ◽

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Food Samples ◽

And Staphylococcus Aureus

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Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

European Food Research and Technology ◽

10.1007/s00217-012-1665-3 ◽

2012 ◽

Vol 234 (4) ◽

pp. 571-580 ◽

Cited By ~ 24

Author(s):

Alejandro Garrido ◽

María-José Chapela ◽

Belén Román ◽

Martiña Ferreira ◽

Jorge Lago ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Salmonella Enterica ◽

Shigella Flexneri ◽

Simultaneous Detection ◽

Food Samples ◽

Processed Food ◽

Pcr Method

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Nonaplex real-time PCR detection of Listeria monocytogenes, Campylobacter, Salmonella and enteropathogene E. coli after universal enrichment in food samples

European Food Research and Technology ◽

10.1007/s00217-013-1994-x ◽

2013 ◽

Vol 237 (3) ◽

pp. 315-322 ◽

Cited By ~ 5

Author(s):

René Köppel ◽

Agne Rüegg Kuslyte ◽

Ingrid Tolido ◽

Jürg Schmid ◽

Gabriela Marti

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Food Samples ◽

Pcr Detection ◽

E Coli ◽

Universal Enrichment

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Enrichment and DNA Extraction Protocols for the Simultaneous Detection of Salmonella and Listeria monocytogenes in Raw Sausage Meat with Multiplex Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-67.1.189 ◽

2004 ◽

Vol 67 (1) ◽

pp. 189-192 ◽

Cited By ~ 58

Author(s):

XIAOWEN WANG ◽

NARAYANAN JOTHIKUMAR ◽

MANSEL W. GRIFFITHS

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Meat Sample ◽

Extraction And Purification ◽

Pcr Products ◽

Novel Method ◽

Hlya Gene

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes, and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-122 ◽

2016 ◽

Vol 79 (1) ◽

pp. 138-143 ◽

Cited By ~ 2

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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