Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Vantarakis, Apostolos; Komninou, G.; Venieri, Danae; Papapetropoulou, M.

doi:10.1046/j.1365-2672.2000.00797.x

Cited by 53 publications

(39 citation statements)

References 19 publications

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“…2 a and b). The results obtained here are in good accordance with the results obtained by MAHON et al (1994), SOUMET et al (1999) and VANTARAKIS et al (2000). Multiplex PCR proved to be a sensitive and a specific method with a superior ability to detect Salmonella enterica in the presence of other competing bacteria, a food sample that is microbiologically contaminated may contain numerous other bacterial species (WANG et al 1997).…”

Section: Discussionsupporting

confidence: 94%

“…Such protocols use different PCR cycler machines, annealing temperatures, MgCl 2 concentrations and buffer systems (WANG et al 1997). In order to reach a reliable PCR system for the detection of all Salmonella enterica serovars, multiplex PCR (m-PCR) appears as a method of choice to amplify several signature-sequences in the same reaction tube (VANTARAKIS et al 2000). This will ensure consistent detection and high specificity and sensitivity.…”

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Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products

Malkawi¹,

Gharaibeh²

2003

J. Basic Microbiol.

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Three sets of known Salmonella enterica-specific primers were used collectively, for the first time, to evaluate the use of multiplex polymerase chain reaction (m-PCR) as a diagnostic tool to detect Salmonella enterica in naturally contaminated meat and poultry products. For this purpose a total of 300 samples representing the most frequently used fresh and frozen meat (beef and lamb) and poultry (chicken) products (whole, cut, ground, and processed) were collected from eight locations within Irbid city (Jordan). After an enrichment step, DNA was extracted directly from each food sample and amplified using six Salmonella enterica specific primers. Samples were also analyzed using conventional microbiological methods for the confirmation of Salmonella enterica presence. Out of 300 samples, 93 samples were positive by m-PCR. On the other hand, 67 samples were positive by conventional microbiological methods and only 26 samples were positive by m-PCR alone. The primer pairs, used here, proved to be highly specific for Salmonella enterica. Using m-PCR, Salmonella enterica detection could be achieved easily and the results had been confirmed effortlessly within a short period of time (24-36 hours) compared to 3-8 days for the conventional microbiological methods. Our findings emphasize the value of using a combination of specific primer pairs instead of a single primer pair in the detection process to minimize any chance for any inherent experimental or natural error.

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Section: Discussionsupporting

confidence: 94%

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confidence: 99%

Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products

Malkawi¹,

Gharaibeh²

2003

J. Basic Microbiol.

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“…Traditional methods of diagnosing bacterial infections using culture techniques require several days to arrive at a definitive diagnosis, resulting in delayed implementation of control measures and increased potential for spreading of disease. Because PCR can target unique genetic sequences of microorganisms, PCR-based nucleic acid amplification techniques have gained recognition as rapid, sensitive, and specific methods for detection of disease-causing pathogens in various aquaculture species (Vantarakis et al 2000, Del Cero et al 2002, Bader et al 2003, Bilodeau et al 2003. When multiple bacterial pathogens are likely to occur, as in the aquatic environment, amplification of multiple target genes in a single reaction mixture is possible with the multiplex PCR (m-PCR) method (Brasher et al 1998, Del Cero et al 2002, Panicker et al 2004, thus reducing cost, time and effort without compromising the test utility.…”

Section: Introductionmentioning

confidence: 99%

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Panangala

Shoemaker²,

Santen³

et al. 2007

Dis. Aquat. Org.

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A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 × 10 2 and 2.5 × 10 5 cells g -1 of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the timeconsuming traditional bacteriological culture techniques. KEY WORDS: Multiplex-PCR · Fish · Edwardsiella · Flavobacterium · AeromonasResale or republication not permitted without written consent of the publisher Dis Aquat Org 74: 199-208, 2007 most important bacterial diseases affecting the aquaculture industry in the USA (United States Department of Agriculture 2003). The prevalence and impact of septicemic disease in fish caused by Aeromonas hydrophila (referred to as motile aeromonad septicemia, MSA), is less known. However, A. hydrophila is ubiquitous in aquatic ecosystems (Holmes et al. 1996) and recent studies have shown that catfish latently infected with A. hydrophila (carriers) could shed the organism when co-infected with Edwardsiella ictaluri (Nusbaum & Morrison 2002). Since all 3 species of bacteria (Flavobacterium columnare, E. ictaluri, and A. hydrophila) are ubiquitous in the aquatic environment and fish are reared in extensive pond acreages with high stocking densities (20 000 to 30 000 fish ha -1 ), the occurrence of coinfections or multiple infections in the same host should not be overlooked (Jack et al. 1992). Traditional methods of diagnosing bacterial infections using culture techniques require several days to arrive at a definitive diagnosis, resulting in delayed implementation of control measures and increased potential for spreading of disease. Because PCR can target unique genetic sequences of microorganisms, PCR-based nucleic acid amplification techniques have gained recognition as rapid, sensitive, and specific methods for detection of disease-causing pathogens in various aquaculture species (Vantarakis et al. 2000, Del Cero e...

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“…The presence of Vibrio and other potential bacterial pathogens such as Escherichia 28 coli, Salmonella and Shigella have been detected in cultivated mussels at retail markets 29 [27,28,29]. Some of these bacteria form part of the natural marine microflora and may be 30 accumulated by shellfish during feeding [30].…”

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confidence: 99%

Elevated concentrations of oxygen on the stability of live mussel stored refrigerated

Pastoriza

Bernárdez

Sampedro

et al. 2004

European Food Research and Technology

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Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Cited by 53 publications

References 19 publications

Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products

Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Elevated concentrations of oxygen on the stability of live mussel stored refrigerated

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