Evaluation of Most-Probable-Number–PCR Method with Internal Amplification Control for the Counting of Total and Pathogenic Vibrio parahaemolyticus in Frozen Shrimps

2012 ◽  
Vol 75 (1) ◽  
pp. 150-153 ◽  
Author(s):  
S. COPIN ◽  
A. ROBERT-PILLOT ◽  
P. MALLE ◽  
M. L. QUILICI ◽  
M. GAY
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Direct Detection ◽  
Selective Medium ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Probable Number ◽  
Pathogenic Vibrio ◽  
Biochemical Identification ◽  
Pcr Method ◽  
Frozen Shrimp

The most-probable-number (MPN) method is often time-consuming for the isolation, detection, and quantification of Vibrio parahaemolyticus from natural sources. MPN counting of V. parahaemolyticus bacteria usually involves the isolation of typical V. parahaemolyticus colonies on selective medium, with subsequent confirmation by biochemical identification. In this study, we evaluated the use of a PCR on MPN enrichment cultures (MPN-PCR) for the direct detection of total and pathogenic V. parahaemolyticus cells in frozen shrimp. This reaction targeted the R72H, tdh, and trh sequences. An internal amplification control was added to the samples before R72H amplification. There was an excellent correlation between the results of the two methods for artificially inoculated and natural shrimp samples. Of 36 natural samples, 28 tested positive for the presence of V. parahaemolyticus, with an MPN value of 2 × 10−1 to 9.2 × 101 per g. No pathogenic V. parahaemolyticus cells were detected. The test had a detection limit of one V. parahaemolyticus organism per g and was completed within two working days. These results support the use of the combination of PCR with MPN for the detection of total or potentially pathogenic V. parahaemolyticus cells in frozen shrimp.

scholarly journals Detection and Quantification of Total and Pathogenic Vibrio parahaemolyticus in Anadara subcrenata in the Zhoushan Archipelago

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Bing Wu ◽  
Hongxia Gong ◽  
Hui Zhang ◽  
Jiabei Chen ◽  
Hongling Wang
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Most Probable Number ◽  
Probable Number ◽  
Retail Markets ◽  
Pathogenic Vibrio ◽  
Food Borne ◽  
Positive Rate ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Zhoushan Archipelago

This study aimed to investigate the prevalence of total and pathogenic Vibrio parahaemolyticus in Anadara subcrenata sampled from aquafarms and retail markets in the Zhoushan islands during June 2013 to March 2015, using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 265 (83.86%) samples with the density 0.3 to 2400 MPN/g. In total, 30.70% and 17.41% of the samples exceeded 100 MPN/g and 1,000 MPN/g, respectively. Both highest positive rate (98.99%) and highest prevalence (median = 210.0 MPN/g) were recorded in summer. Samples from aquafarms had a higher positive rate and median than those from retail markets. Pathogenic V. parahaemolyticus was detected both in aquafarms and retail markets in all seasons but not in winter. Among the 265 tlh-positive samples, 20 (7.55%) of the samples harbored tdh, and 5 (1.89%) of the samples harbored both tdh and trh. These results indicate that the Zhoushan archipelago is severely contaminated with V. parahaemolyticus in Anadara subcrenata; these results are applicable in risk assessment and to control the risk of food-borne disease caused by V. parahaemolyticus.

scholarly journals Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan

2003 ◽  
Vol 69 (7) ◽  
pp. 3883-3891 ◽  
Author(s):  
Yukiko Hara-Kudo ◽  
Kanji Sugiyama ◽  
Mitsuaki Nishibuchi ◽  
Ashrafuzzaman Chowdhury ◽  
Jun Yatsuyanagi ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Most Probable Number ◽  
Asian Countries ◽  
Probable Number ◽  
Specific Pcr ◽  
Thermostable Direct Hemolysin ◽  
Chromogenic Agar ◽  
Pcr Method ◽  
Arbitrarily Primed Pcr

ABSTRACT Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.

scholarly journals Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

2007 ◽  
Vol 73 (18) ◽  
pp. 5840-5847 ◽  
Author(s):  
Jessica L. Nordstrom ◽  
Michael C. L. Vickery ◽  
George M. Blackstone ◽  
Shelley L. Murray ◽  
Angelo DePaola
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Specific Marker ◽  
Pcr Assay ◽  
Probable Number ◽  
The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

Development of a Quantitative Real-Time PCR Method for Estimation of the Total Number of Vibrio parahaemolyticus in Contaminated Shellfish and Seawater

2005 ◽  
Vol 68 (5) ◽  
pp. 1083-1088 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
YOSHITO IWADE ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
Vibrio Parahaemolyticus ◽  
Plate Count ◽  
Most Probable Number ◽  
Cycle Number ◽  
Probable Number ◽  
The Real ◽  
Pcr Method

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 101 to 107 CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.

Pathogenic Vibrio Species in Dutch Shellfish Destined for Direct Human Consumption

2010 ◽  
Vol 73 (4) ◽  
pp. 734-738 ◽  
Author(s):  
FRANCISKA M. SCHETS ◽  
HAROLD H. J. L. van den BERG ◽  
SASKIA A. RUTJES ◽  
ANA MARIA de RODA HUSMAN
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Vibrio Species ◽  
Most Probable Number ◽  
Human Consumption ◽  
Gastrointestinal Infections ◽  
Probable Number ◽  
The North ◽  
Pathogenic Vibrio ◽  
The North Sea ◽  
Production Areas

Vibrio parahaemolyticus is a common cause of shellfish-related gastroenteritis all over the world. V. parahaemolyticus and Vibrio alginolyticus have previously been detected in water samples from the Oosterschelde, a large inlet on the North Sea, which is used for both recreational purposes and shellfish production. In 2006, oysters (Crassostrea gigas) from a noncommercial oyster bed in the Oosterschelde and oysters bought in Dutch fish shops were tested for the presence of pathogenic Vibrio species; in 2007 and 2008, oysters (C. gigas) and mussels (Mytilus edulis) from Oosterschelde production areas were examined. Total Vibrio numbers were related to water temperatures to study joint patterns. Vibrio was found in oysters and mussels from the production areas, and levels ranged from 6 to 622 most probable number (MPN) per g in oysters and 6 to 62 MPN/g in mussels. Vibrio levels in oysters from fish shops were 231 to &gt;333 MPN/g, whereas levels in noncommercial oysters ranged from 231 to &gt;2,398 MPN/g. About 80% of the isolated strains were V. alginolyticus, and approximately 10% were identified as V. parahaemolyticus. Vibrio counts in shellfish samples increased with increasing water temperature and declined when water temperatures dropped; Vibrio was not detected when water temperatures declined to &lt;13.5°C. Based on the obtained results and the known high V. parahaemolyticus dose (&lt;104 cells per serving of oysters) required for infection, it is concluded that the risk of gastrointestinal infections with V. parahaemolyticus through consumption of shellfish from the Oosterschelde production sites is presumably low.

scholarly journals Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Salina Parveen ◽  
John Jacobs ◽  
Gulnihal Ozbay ◽  
Lathadevi K. Chintapenta ◽  
Esam Almuhaideb ◽  
...  
Keyword(s):  
Chesapeake Bay ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Seawater Temperature ◽  
False Negative ◽  
Delaware Bay ◽  
Most Probable Number ◽  
Probable Number ◽  
Content Type ◽  
Pathogenic Vibrio

ABSTRACT Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number–PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+) and pathogenic (tdh+ and trh+) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae. DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+ V. parahaemolyticus and vvhA+ V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+ V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+ V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay. IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

An improved selection technique and medium for the isolation and enumeration of Azospirillum brasilense

1985 ◽  
Vol 31 (10) ◽  
pp. 947-952 ◽  
Author(s):  
Yoav Bashan ◽  
Hanna Levanony
Keyword(s):  
Azospirillum Brasilense ◽  
Sodium Deoxycholate ◽  
Selective Medium ◽  
Most Probable Number ◽  
Streptomycin Sulphate ◽  
Triphenyltetrazolium Chloride ◽  
Wheat Roots ◽  
Semisolid Medium ◽  
Probable Number ◽  
Selection Technique

An improved selection technique for isolation and enumeration of Azospirillum brasilense was developed. The technique is based on successive liquid enrichments in nitrogen-free semisolid medium supplemented with streptomycin, followed by the most probable number counting method and verification on a selective medium. The latter is based on Okon's nitrogen-free medium supplemented with cycloheximide (250 mg/L), streptomycin sulphate (200 mg/L), sodium deoxycholate (200 mg/L), 2,3,5-triphenyltetrazolium chloride (15 mg/L), and Congo red (1000 mg/L). This medium was found to be superior to other available diagnostic media. The technique was readily applied to detect and count A. brasilense Cd in inoculated wheat roots.

Selectivity and Specificity of a Chromogenic Medium for Detecting Vibrio parahaemolyticus†

2005 ◽  
Vol 68 (7) ◽  
pp. 1454-1456 ◽  
Author(s):  
YI-CHENG SU ◽  
JINGYUN DUAN ◽  
WEN-HSIN WU
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Bile Salts ◽  
Vibrio Vulnificus ◽  
Enterobacter Cloacae ◽  
Most Probable Number ◽  
Chromogenic Medium ◽  
Probable Number ◽  
Vibrio Fluvialis ◽  
Vibrio Mimicus ◽  
Vibrio Spp

The thiosulfate–citrate–bile salts–sucrose agar (TCBS) used in the most-probable-number method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus. This study examined the selectivity and specificity of Bio-Chrome Vibrio medium (BCVM), a chromogenic medium that detects V. parahaemolyticus on the basis of the formation of distinct purple colonies on the medium. A panel consisting of 221 strains of bacteria, including 179 Vibrio spp. and 42 non-Vibrio spp., were examined for their ability to grow and produce colored colonies on BCVM. Growth of Salmonella, Shigella, Escherichia coli, Enterobacter cloacae, Yersinia enterocolitica, and Aeromonas was inhibited by both BCVM and TCBS. All 148 strains of V. parahaemolyticus grew on BCVM, and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of Vibrio fluvialis, were either unable to grow or produced blue-green or white colonies on BCVM. Bio-Chrome Vibrio medium was capable of differentiating V. parahaemolyticus from other species, including V. vulnificus and V. mimicus. Further studies are needed to evaluate the sensitivity and specificity of BCVM for detecting V. parahaemolyticus in foods.

scholarly journals Long-Term Study of Vibrio parahaemolyticus Prevalence and Distribution in New Zealand Shellfish

2015 ◽  
Vol 81 (7) ◽  
pp. 2320-2327 ◽  
Author(s):  
C. D. Cruz ◽  
D. Hedderley ◽  
G. C. Fletcher
Keyword(s):  
New Zealand ◽  
Vibrio Parahaemolyticus ◽  
Most Probable Number ◽  
Pcr Analysis ◽  
Potential Hazard ◽  
Probable Number ◽  
Content Type ◽  
The North ◽  
Long Term Study ◽  
Food Borne

ABSTRACTThe food-borne pathogenVibrio parahaemolyticushas been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence ofV. parahaemolyticusin NZ oysters and Greenshell mussels and the prevalence ofV. parahaemolyticustdhandtrhstrains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. TotalV. parahaemolyticusnumbers and the presence of pathogenic genestdhandtrhwere determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ,V. parahaemolyticuswas detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers ofV. parahaemolyticustdhandtrhstrains were low, with just 3/215 Pacific oyster samples carrying thetdhgene.V. parahaemolyticusorganisms carryingtdhandtrhwere not detected in South Island samples, andV. parahaemolyticuswas detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers ofV. parahaemolyticusorganisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers ofV. parahaemolyticusorganisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the totalV. parahaemolyticusnumbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.

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