scholarly journals Improvement of the quantitation method for the tdh+ Vibrio parahaemolyticus in molluscan shellfish based on most-probable- number, immunomagnetic separation, and loop-mediated isothermal amplification

2015 ◽  
Vol 6 ◽  
Author(s):  
Oscar Escalante-Maldonado ◽  
Ahmad Y. Kayali ◽  
Wataru Yamazaki ◽  
Varaporn Vuddhakul ◽  
Yoshitsugu Nakaguchi ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Immunomagnetic Separation ◽  
Isothermal Amplification ◽  
Most Probable Number ◽  
Probable Number ◽  
Loop Mediated Isothermal Amplification ◽  
Quantitation Method

scholarly journals Most-Probable-Number Loop-Mediated Isothermal Amplification–Based Procedure Enhanced with K Antigen–Specific Immunomagnetic Separation for Quantifying tdh+ Vibrio parahaemolyticus in Molluscan Shellfish

2014 ◽  
Vol 77 (7) ◽  
pp. 1078-1085 ◽  
Author(s):  
NATSUKO TANAKA ◽  
YOSHITO IWADE ◽  
WATARU YAMAZAKI ◽  
FUMIO GONDAIRA ◽  
VARAPORN VUDDHAKUL ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Lamp Assay ◽  
Immunomagnetic Separation ◽  
Microtiter Plate ◽  
Isothermal Amplification ◽  
Most Probable Number ◽  
Probable Number ◽  
Loop Mediated Isothermal Amplification ◽  
Wide Range ◽  
Thermostable Direct Hemolysin

Although thermostable direct hemolysin–producing (tdh+) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh+V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)–based procedure designated A-IS1-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate–based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh+V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (<3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh+V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh+V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh+V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS1-LAMP procedure can be used by any health authority in the world to measure the tdh+V. parahaemolyticus levels in shellfish products.

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Sensitive Detection of Vibrio parahaemolyticus

2011 ◽  
Vol 74 (9) ◽  
pp. 1462-1467 ◽  
Author(s):  
JIRO NEMOTO ◽  
MASANARI IKEDO ◽  
TADASHI KOJIMA ◽  
TAKAYOSHI MOMODA ◽  
HIROTAKA KONUMA ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Lamp Assay ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Most Probable Number ◽  
Lamp Method ◽  
Probable Number ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Rpod Gene

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.

scholarly journals Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in < 25 min

2017 ◽  
Vol 132 ◽  
pp. 27-33 ◽  
Author(s):  
Farhan Ahmad ◽  
Robert D. Stedtfeld ◽  
Hassan Waseem ◽  
Maggie R. Williams ◽  
Alison M. Cupples ◽  
...  
Keyword(s):  
Isothermal Amplification ◽  
Most Probable Number ◽  
Waterborne Pathogens ◽  
Probable Number ◽  
Loop Mediated Isothermal Amplification

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.

Vibrio parahaemolyticus in Long Island Oysters

1982 ◽  
Vol 45 (2) ◽  
pp. 150-151 ◽  
Author(s):  
ANTHONY A. TEPEDINO
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Long Island ◽  
Most Probable Number ◽  
Ileal Loop ◽  
Probable Number ◽  
Number Range

Twelve of 36 samples of Long Island oysters were found to contain Vibrio parahaemolyticus with a most probable number range of 3.6 to 23 organisms/g. Six of 10 isolates tested were weakly Kanagawa positive. None was pathogenic by the rabbit ileal loop test.

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