Identification of Listeria monocytogenes on Green Mussels (Perna viridis) and Cockle Shell (Anadara granosa)

Green mussel (Perna viridis) and cockle shell (Anadara granosa) are one of many sources of animal protein which is many cultivated in Indonesia because their price is relatively affordable. This study was conducted to identify the presence of Listeria monocytogenes in 27 samples of green mussels and 3 samples of cockle shells using real-time Polymerase Chain Reaction (real-time PCR) and biochemical methods. The target gene for amplification in real-time PCR was an hlyA gene because this gene was a determinant of virulence genes that produce listeriolysin O. Primers used in this study were forward primer DG69 (GTG CCG GGT AAA AGA CCA TA) and reverse primer DG74 (CGC CAC TGA GAT ACT AT) and fluorescence signals indicator using SYBR Green I. The results of analysis using real-time PCR were negative Listeria monocytogenes in all samples, while using biochemical methods there was one of 30 samples contaminated by Listeria welshimeri.

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Identification of Listeria monocytogenes on Green Mussels and Cockle Shell

Jurnal Pengolahan Hasil Perikanan Indonesia ◽

10.17844/jphpi.v19i3.15110 ◽

2017 ◽

Vol 19 (3) ◽

pp. 329

Author(s):

Winiati Puji Rahayu ◽

Ristia Rinanti ◽

Siti Nurjanah ◽

Caecillia Chrismie Nurwitri

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Perna Viridis ◽

Listeriolysin O ◽

Biochemical Methods ◽

Reverse Primer ◽

Many Sources ◽

Hlya Gene ◽

Cockle Shell

Abstract Green mussel (Perna viridis) and cockle shell (Anadara granosa) are one of many sources of animal protein which is many cultivated in Indonesia because their price is relatively affordable. This study was conducted to identify the presence of Listeria monocytogenes in 27 samples of green mussels and 3 samples of cockle shells using real-time Polymerase Chain Reaction (real-time PCR) and biochemical methods. The target gene for amplification in real-time PCR was an hlyA gene because this gene was a determinant of virulence genes that produce listeriolysin O. Primers used in this study were forward primer DG69 (GTG CCG GGT AAA AGA CCA TA) and reverse primer DG74 (CGC CAC TGA GAT ACT AT) and fluorescence signals indicator using SYBR Green I. The results of analysis using real-time PCR were negative Listeria monocytogenes in all samples, while using biochemical methods there was one of 30 samples contaminated by Listeria welshimeri.

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

ZANCO Journal of Pure and Applied Sciences ◽

10.21271/zjpas.29.1.15 ◽

2017 ◽

Vol 29 (1) ◽

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Food Samples ◽

Hlya Gene

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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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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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Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

Applied and Environmental Microbiology ◽

10.1128/aem.66.10.4266-4271.2000 ◽

2000 ◽

Vol 66 (10) ◽

pp. 4266-4271 ◽

Cited By ~ 187

Author(s):

Hege Karin Nogva ◽

Knut Rudi ◽

Kristine Naterstad ◽

Askild Holck ◽

Dag Lillehaug

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Direct Detection ◽

Skim Milk ◽

Raw Milk ◽

Magnetic Bead ◽

Quantitative Detection ◽

Listeriolysin O ◽

Pure Cultures

ABSTRACT PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenesbased on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all otherListeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

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