Rapid Quantification of Viable
            <i>Campylobacter</i>
            Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment

Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Tina Beck; Christensen, Laurids Siig; Olsen, John Elmerdahl; Hoorfar, Jeffrey

doi:10.1128/aem.00411-10

Cited by 155 publications

(146 citation statements)

References 37 publications

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“…PMA is an intercalating DNA agent which enters dead cells and binds to DNA, inhibiting subsequent PCR amplification and thereby ensuring quantification of viable bacteria (21). PMA has been used in combination with several real-time PCR methods for quantification of viable bacteria in food, e.g., Campylobacter (22), Listeria monocytogenes (23), Brochothrix thermosphacta (24,25), Vibrio parahaemolyticus (26,27), and Escherichia coli O157:H7 (28).…”

mentioning

confidence: 99%

Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Macé

Mamlouk

Chipchakova

et al. 2013

Appl Environ Microbiol

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A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmospherepacked salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R 2 of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log CFU/g. A correlation coefficient (R 2 ) of 0.963 was obtained between the PCR method and classic enumeration on MA, followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rRNA gene sequencing). A good correlation with an R 2 of 0.940 was found between the new PCR method and an available specific conductance method for P. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viable P. phosphoreum bacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism. Modified-atmosphere-packed (MAP) fresh fish is increasingly popular in Europe and widely sold in supermarkets as a chilled product. Compared to aerobically stored fresh fish, this kind of packaging, with typical headspace CO 2 concentrations of 20 to 60%, modifies the dominating microbiota mainly by reducing growth of aerobic Gram-negative bacteria like Pseudomonas (1, 2). This results in an extended shelf life which facilitates the chilled distribution and marketing of fresh MAP fish. However, this packaging allows the growth of CO 2 -resistant bacteria, including Gram-positive lactic acid bacteria and the Gram-negative bacterium Photobacterium phosphoreum (2-4). The latter has been identified as the specific spoilage organism (SSO) responsible for trimethylamine production and sensory spoilage of MAP cod (5, 6) and as the main spoilage bacterial species of several chilled marine fish, including cod, garfish, halibut, saithe, salmon, and shrimp (7-15).Multilocus analysis, based on 16S rRNA and gyrB and luxABFE genes, divided strains originally isolated and identified as P. phosphoreum into three distinct clades of P. phosphoreum, Photobacterium iliopiscarium, and Photobacterium kishitanii (16,17). The members of the P. phosphoreum species group have been reported as important for spoilage of raw MAP fish, and they include both luminous and nonluminous variants (5, 18). It is therefore relevant to detect and enumerate this s...

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mentioning

confidence: 99%

Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Macé

Mamlouk

Chipchakova

et al. 2013

Appl Environ Microbiol

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“…PMA intercalates DNA from dead cells with compromised cell membranes, upon visible light exposure it binds covalently to DNA thus inhibiting PCR reaction. PMA treatment combined with real-time PCR has been successively used for the quantification of viable cells of foodborne pathogenic microorganisms such as Listeria monocytogenes (Pan & Breidt, 2007), E. coli 0157:H7 (Elizaquível, S anchez, Selma, & Aznar, 2012) and Campylobacter jejuni (Josefsen et al, 2010), but also for viable cells of the biocontrol agent Pantoea agglomerans CPA-2, effective against the major postharvest diseases of pome and citrus fruits . Thus, in the future it will be interesting to verify the feasibility of the introduction of a pre-treatment with PMA to Pseudomonas-based BCAs before real-time PCR quantification.…”

Section: Future Perspectivesmentioning

confidence: 99%

Quantitative real-time PCR and high-resolution melting (HRM) analysis for strain-specific monitoring of fluorescent pseudomonads used as biocontrol agents against soil-borne pathogens of food crops

Martini

Moruzzi

Ermacora

et al. 2015

Trends in Food Science & Technology

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“…PCR-based detection of pathogens has therefore become increasingly popular in recent times. Effective PCR-detection assays have been successfully designed and implemented for a broad range of these bacterial food-borne pathogens such as Salmonella, Campylobacter, Bacillus cereus, pathogenic Escherichia coli (EHEC) and others (Anderson et al, 2010, Lehmann et al, 2010, Josefsen et al, 2010, Fratamico et al, 2011, Wang et al, 2011.…”

Section: Molecular Biology Tools For Detection Of Foodborne Pathogensmentioning

confidence: 99%

“…3). In a recent study by Josefsen et al, 2010, a CFU-based standard curve was utilized in the quantitative determination of Campylobacter in chicken rinse (Josefsen et al, 2010). In this work, the quantification method was compared with culture-based enumeration on 50 naturally infected chickens.…”

Section: Pcr-based Food -Borne Pathogen (Bacteria) Detectionmentioning

confidence: 99%

“…PMA possesses an azide group whch permits cross-linking of the dye to DNA after exposure to strong visible light. When the PMA-treated cells are subjected to DNA extraction procedures and subsequently PCR for detection of the bacteria of interest, a reduction in the number of detectable bacteria is often observed with PMA-treated cells (Josefsen et al, 2010). The PMA approach is currently being developed and validated in our laboratory for the reliable identification and quantification of viable and live bacterial pathogens in various food matrices.…”

Section: Pcr-based Food -Borne Pathogen (Bacteria) Detectionmentioning

confidence: 99%

See 1 more Smart Citation

The Application of PCR-Based Methods in Food Control Agencies – A Review

Iwobi¹,

Huber²,

Busch³

2012

Polymerase Chain Reaction

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Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment

Cited by 155 publications

References 37 publications

Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Quantitative real-time PCR and high-resolution melting (HRM) analysis for strain-specific monitoring of fluorescent pseudomonads used as biocontrol agents against soil-borne pathogens of food crops

The Application of PCR-Based Methods in Food Control Agencies – A Review

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