Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR

Zhu, Rongshu; Li, Tuoping; Jia, Yingmin; Li-feng, Song

doi:10.1016/j.mimet.2012.05.019

Cited by 44 publications

(49 citation statements)

References 29 publications

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“…In recent years, culture-independent molecular methods like real-time PCR have been increasingly applied to detect and quantify target microorganisms in food (36,37). Several real-time PCR methods have been developed to enumerate or detect pathogenic bacteria in seafood products, including Vibrio parahaemolyticus (26,38) and mesophilic Gram-negative histamine-producing bacteria (39,40). Spoilage bacteria like Pseudomonas (41) and Brochothrix thermosphacta (24) have also been enumerated by these methods.…”

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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“…PMA is a DNA intercalating reagent, which only penetrates the cell membranes of dead cells and covalently binds to DNA through photolysis (Nocker and Cheung Camper 2006;Pan and Breidt 2007;Luo et al 2010;Schnetzinger et al 2013;Macé et al 2013;Salam et al 2014). This reaction inhibits subsequent PCR amplification, thereby ensuring quantification of viable bacteria (Nocker and Cheung Camper 2006;Pan and Breidt 2007;Zhu et al 2012;Macé et al 2013;Liu and Mustapha 2014;Salam et al 2014). This reagent has been used in combination with several real-time PCR methods for quantification of viable pathogens in food, such as V. parahaemolyticus (Zhu et al 2012), L. monocytogenes (Pan and Breidt 2007), Campylobacter (Josefsen et al 2010), Salmonella spp.…”

Section: Introductionmentioning

confidence: 99%

“…This reaction inhibits subsequent PCR amplification, thereby ensuring quantification of viable bacteria (Nocker and Cheung Camper 2006;Pan and Breidt 2007;Zhu et al 2012;Macé et al 2013;Liu and Mustapha 2014;Salam et al 2014). This reagent has been used in combination with several real-time PCR methods for quantification of viable pathogens in food, such as V. parahaemolyticus (Zhu et al 2012), L. monocytogenes (Pan and Breidt 2007), Campylobacter (Josefsen et al 2010), Salmonella spp. (Barbau-Piednoir et al 2014), and Escherichia coli (Liu and Mustapha 2014).…”

Section: Introductionmentioning

confidence: 99%

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Zhang

Liu

Lou

et al. 2015

Appl Microbiol Biotechnol

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A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10(8) CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10(1)-10(2) CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

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“…Efficient amounts of sample DNA of high quality is of critical importance for molecular assays. So far, different methods have been used to extract the DNA samples, including commercial kits [19,21,22], the phenol-chloroform method [15,23] and the boiling lysis method [10,24,25].…”

Section: Introductionmentioning

confidence: 99%

http://www.omicsonline.org/open-access/overcoming-pseudomonas-aeruginosa-resistance-caused-by-glycocalyx-with-tobracef-1948-5948.1000145.php?aid=25925

Dai¹

2014

J Microb Biochem Technol

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Oysters are filter feeders that bioaccumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic DNA (Allrun, Shanghai, China), the MiniBEST Bacterial Genomic DNA Extraction kits (Takara, Dalian, China), and the phenol-chloroform and boiling lysis methods. The concentration of the genomic DNA was measured using a spectrophotometer. The purity of the genomic DNA was evaluated by PCR amplification of 16S rDNA followed by determining the cloning efficiency of the amplicon into the pMD19-T vector. Furthermore, the bacterial DNA quality was also evaluated by PCR assays using a pair of species-specific primers for Vibrio parahaemolyticus. Our results showed that the two commercial kits produced the highest purity of DNA, but with the lowest yields. The phenol-chloroform method produced the highest yield although it was time-consuming. The boiling lysis method was simple and cost effective; however, it was only suitable to isolate genomic DNA from bacteria present in retail samples following an enrichment step. The two commercial kits were good candidates for genomic DNA extraction from retail oyster tissues without enrichment.

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Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR

Cited by 44 publications

References 29 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

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

http://www.omicsonline.org/open-access/overcoming-pseudomonas-aeruginosa-resistance-caused-by-glycocalyx-with-tobracef-1948-5948.1000145.php?aid=25925

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