Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Robert-Pillot, Annick; Copin, S.; Malle, Pierre; Quilici, Marie‐Laure

doi:10.1016/j.ijfoodmicro.2010.08.016

Cited by 38 publications

(14 citation statements)

References 47 publications

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“…To improve the sensitivity of the new real-time PCR method, an enrichment step in broth, prior to the PCR assay, could be used, e.g., in the same way as applied for the available conductance method for sensitive enumeration of P. phosphoreum (19). Enrichment steps in the broth or food samples have already been used to enhance the sensitivity of real-time PCR methods, for both qualitative and quantitative detection of the target bacteria (27,44,51). In fact, we tested this approach by using quantitative enrichment in Photobacterium phosphoreum differential medium (PPDM) at 15°C (19).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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“…Utilization of the enrichment-PCR method 27,28) also requires more time than the newly developed assay of this study, as additional gel-run and detection procedures are required after PCR step which is avoided by the use of real-time turbidimeter in the LAMP assay. A recent study has reported successful utilization of real-time PCR method after a 6 h enrichment period in detecting less than 5 V. parahaemolyticus cells/g of seafood, 29) however, this method is more expensive and complex, and requires more time than the LAMP assay of the present study. In addition to rapid detection, the LAMP assay has other advantages, such as quantitative measurement, a lower contamination rate, higher sensitivity and specificity, and easy standardization.…”

Section: Specificity and Sensitivity Of Lamp Detectionmentioning

confidence: 65%

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

Neogi

et al. 2015

Biol. Pharm. Bull.

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The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n 61) but none of the non-target strains (n 34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n 70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains.Key words Vibrio parahaemolyticus; loop-mediated isothermal amplification; rapid detection; enrichment culture; seafood Vibrio parahaemolyticus is one of the most important seafood-borne pathogen causing gastrointestinal disorders to humans. This halophilic Gram-negative bacterium was first identified as a causative agent of human gastroenteritis in Japan in 1950.1) However, V. parahaemolyticus can occur ubiquitously in the brackish coastal environment of most continents and infections associated with seafood contaminated by this pathogen have occurred throughout the world.2-4) Particularly, the worldwide spread of the V. parahaemolyticus O3 : K6 serotype after its emergence in Asia in 1995, it has become very important to identify this pathogenic species in a rapid and sensitive manner.5) At present, the outbreak of V. parahaemolyticus infections is a significant public health concern in many countries including China, Japan and U.S.A. 6,7)Seafood is very popular in many countries, and simple and specific identification of a pathogen from seafood samples is essential for taking preventive and curative measures. Conventional methods for the diagnosis of V. parahaemolyticus include cultivation of bacteria on selective media followed by biochemical tests. However, the traditional phenotypic identification is problematic, it is very time-consuming requiring 3-7 d and not highly specific because several Vibrio species display similar biochemical characteristics, which make it difficult for rapid identification of V. parahaemolyticus. 8) To circumvent this problem, the identification of V. parahaemolyticus by rapid and specific molecular techniques targeting the genes encoding thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) were developed.9-11) However, these genes are only found in pathogenic strains and most V. parahaemolyticus isolates from the environment sources do not produce TDH or TRH. 3,12) Besides, there are reports of toxigenic factors other than TDH or TRH, e.g., type III secretion system 2, associated with clinical V. parahaemolyticus stra...

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“…Then, a loop of biomass was harvested on plates and subjected to DNA extraction with the DNeasy Blood & Tissue Kit from Qiagen (Courtaboeuf, France). The manufacturer's instructions were then followed except for the final step; RNase from Promega (Charbonnières-les-Bains, France) was added after the lysis step at 56 ∘ C. As advised by a previous study, 30 the final elution with AE buffer was split into two elutions of 50 μL to increase the DNA concentration of the eluate. Finally, DNA concentration was measured with a DS-11 Spectrophotometer from Denovix (Wilmington, NC, USA).…”

Section: Dna Extraction From Pure Culturementioning

confidence: 99%

Monitoring the freshness of fish: development of a qPCR method applied to MAP chilled whiting

et al. 2015

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Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Cited by 38 publications

References 47 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

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

Monitoring the freshness of fish: development of a qPCR method applied to MAP chilled whiting

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