BACKGROUND
vibrio parahaemolyticus is a marine seafoodborne pathogen causing gastrointestinal disorders in humans  <cit> .
thermostable direct hemolysin  and tdh-related hemolysin  are known as major virulence determinants of v.parahaemolyticus  <cit> .
this bacterium is widely present in estuarine, marine, and coastal environments throughout the world  <cit> .
therefore, ingestion of raw or undercooked seafood contaminated with v.parahaemolyticus is risk factors in humans  <cit> .
most v.parahaemolyticus isolates from the environment do not produce tdh or trh.
virulent strains of v. parahaemolyticus are usually found together with larger populations of avirulent strains in the environment  <cit> .
the similarity in growth kinetics of the virulent and avirulent strains is a major obstacle for selective detection of virulent strains in seafood.
total v. parahaemolyticus has thus been used as an indicator for control of food contamination toward prevention of infection.
thermolabile hemolysin  has been characterised by taniguchi and colleagues  <cit> , which has been found in all v. parahaemolyticus isolates.
<cit> .
this hemolysin is species specific and not a virulence factor.
this gene is therefore useful target for detection of total v. parahaemolyticus.
detection of total v. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days.
a rapid, reliable and practical assay for the detection of total v. parahaemolyticus has been sought.
several pcr assays offer a more sophisticated approach to the identification of v. parahaemolyticus  <cit> .
although pcr assays provide more rapid identification of v. parahaemolyticus than conventional biochemical-based assays, they require electrophoresis in an agarose gel, which is time-consuming and tedious.
real time pcr assays recently developed for identification of v. parahaemolyticus  <cit>  are rapider than conventional pcr assays due to the detection of fluorescence from amplification.
real-time pcr assay is, however, not routinely used due to the requirement for an expensive thermal cycler with a fluorescence detector.
among other techniques one promising candidate is a novel nucleic acid amplification method termed loop-mediated isothermal amplification   <cit> .
several investigators have developed lamp assays for detection of pathogenic microorganisims  <cit> .
lamp assay is faster and easier to perform than conventional pcr assays, as well as being more specific  <cit> .
furthermore, because the lamp assay synthesizes a large amount of dna, the products can be detected by simple turbidity.
thus, compared to pcr assays, expensive equipment is not necessary to give a high level of precision  <cit> .
these features allow simple, rapid and cost-effective detection  <cit> .
also, the increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of dna synthesized  <cit> .
in addition, the preparation steps of the lamp assay are fewer than with conventional pcr and real-time pcr assays, and lamp assays require less time than those assays  <cit> .
although various lamp assays for the identification of pathogenic organisms have been developed, no assay for the detection of v. parahaemolyticus has been described.
here, we describe a sensitive, rapid and simple lamp assay for the detection of v. parahaemolyticus.
sensitivity was determined in pure cultures and in spiked shrimp samples.
RESULTS
lamp products were detected from all  <dig> v. parahaemolyticus strains.
no lamp products were detected from any of the  <dig> non-parahaemolyticus vibrio and  <dig> non-vibrio strains .
the pcr assay required more than  <dig> h, while the lamp assay was markedly faster, requiring for amplification 13– <dig> min in a single colony on tcbs agar from each of  <dig> v. parahaemolyticus strains and less than  <dig> min in spiked shrimp samples .
the assay required less than  <dig> min and  <dig> min for detection of v. parahaemolyticus in a colony on tcbs agar and in spiked shrimp samples from the beginning of dna extraction to final determination.
a) described in the methods section.
as shown in table  <dig>  sensitivities of the lamp assay for v. parahaemolyticus aq <dig> in pure cultures and in spiked shrimp samples were found to be  <dig>  ×  <dig> cfu per ml .
further, the sensitivity of the lamp assay was 10-fold higher than that of the pcr assay .
the dilutions yielding  <dig>  - <dig>  cfu per reaction showed an increase in turbidity  and was visible as white turbidity but not that of  <dig>  cfu per reaction.
sensitivities determined by the two methods were constantly matched with each other.
when lamp assay performed in triplicate using  <dig> μl templates dna containing  <dig>  cfu per reaction from pure cultures, two of three samples showed positive results, as well as using  <dig> μl templates dna from shrimp samples .
+, triplicate assay showed all positive.
±, triplicate assay showed both positive and negative .
-, triplicate assay showed all negative.
nd, not determined.
discussion
the bacterial culture test for the isolation and identification of v. parahaemolyticus from food samples after enrichment requires 2– <dig> d, with plating onto selective agars, sequential subculture and biochemical characteristic test.
in contrast, the lamp assay was markedly faster.
conventional pcr assay requires 4– <dig> h for amplification, electrophoresis and staining, while the lamp assay requires for dna extraction from samples and amplification less than 40– <dig> min.
further, amplification of the lamp assay could be judged by visual assessment using the naked eye, without the need for electrophoresis.
the lamp assay was more sensitive, rapid and simple than the conventional pcr assay.
therefore, the lamp assay is more effective in detecting v. parahaemolyticus than the conventional pcr assay.
in the preliminary tests, the lamp assay using  <dig>   <dig> and  <dig> μl templates dna from shrimp samples was enough for amplification without any inhibition of the reaction in  <dig> μl reaction mixture volumes.
the pcr assay was, however, shown inhibited results in  <dig> μl reaction mixture volumes.
no pcr products were detected from the pcr reaction mixtures containing  <dig> μl templates from shrimp samples.
slight bands were detected from the mixtures containing  <dig> μl templates.
clear bands were obtained from each of the mixture containing  <dig> μl or  <dig>  μl templates.
it is known that some components of food inhibit dna amplification such as lamp and pcr assays  <cit> .
the results seem to suggest lamp is more resistant than pcr assay to inhibition by shrimp components.
contamination of v. parahaemolyticus in seafood is one of the most important public health hazards  <cit> .
the frequent outbreaks caused by v. parahaemolyticus worldwide highlight the need for control of contamination of v. parahaemolyticus in seafood.
especially, ingestion of fresh raw seafood as sushi and sashimi dishes in japan is a risk factor to humans  <cit> .
the total number of v. parahaemolyticus cells in fish fillets and shellfishes is required below  <dig> cfu/g for raw consumption by law in japan.
to reduce the risk of v. parahaemolyticus infection and to ensure food safety, our lamp assay would be significant in the detection of total v. parahaemolyticus in routine tests and in potentially applying to the most-provable-number method.
rapid, simple and sensitive assay for detection of v. parahaemolyticus by lamp should facilitate the surveillance for control of contamination of v. parahaemolyticus in seafood.
the lamp assay has potential value for rapid and simple screening of total v. parahaemolyticus-contaminated samples before they are consumed.
although the shrimp samples were artificially spiked, we successfully developed the first lamp assay for detection of total v. parahaemolyticus from seafood samples.
development of the lamp assay for detection of virulent strains of v. parahaemolyticus is required, as well as real-time and conventional pcr assays  <cit> .
further studies will be performed for detection of virulent v. parahaemolyticus and evaluation of the assay using a large number of seafood naturally contaminated by v. parahaemolyticus.
CONCLUSIONS
the lamp assay provided markedly more sensitive, simple and rapid detection of v. parahaemolyticus than conventional biochemical and pcr assays.
the lamp assay for detection of v. parahaemolyticus required less than  <dig> min in a colony on tcbs agar and  <dig> min in spiked shrimp samples from the beginning of dna extraction to final determination.
the lamp assay is a powerful tool for the rapid and sensitive detection of v. parahaemolyticus, and will facilitate the surveillance for control of contamination of v. parahaemolyticus in seafood.

 vibrio parahaemolyticus is a marine seafoodborne pathogen causing gastrointestinal disorders in humans. thermostable direct hemolysin and tdh-related hemolysin are known as major virulence determinants of v.parahaemolyticus. this bacterium is widely present in estuarine, marine, and coastal environments throughout the world. therefore, ingestion of raw or undercooked seafood contaminated with v.parahaemolyticus is risk factors in humans. most v.parahaemolyticus isolates from the environment do not produce tdh or trh. virulent strains of v. parahaemolyticus are usually found together with larger populations of avirulent strains in the environment. the similarity in growth kinetics of the virulent and avirulent strains is a major obstacle for selective detection of virulent strains in seafood. total v. parahaemolyticus has thus been used as an indicator for control of food contamination toward prevention of infection. thermolabile hemolysin has been characterised by taniguchi and colleagues, which has been found in all v. parahaemolyticus isolates.. this hemolysin is species specific and not a virulence factor. this gene is therefore useful target for detection of total v. parahaemolyticus. detection of total v. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. a rapid, reliable and practical assay for the detection of total v. parahaemolyticus has been sought. several pcr assays offer a more sophisticated approach to the identification of v. parahaemolyticus. although pcr assays provide more rapid identification of v. parahaemolyticus than conventional biochemical-based assays, they require electrophoresis in an agarose gel, which is time-consuming and tedious. real time pcr assays recently developed for identification of v. parahaemolyticus are rapider than conventional pcr assays due to the detection of fluorescence from amplification. real-time pcr assay is, however, not routinely used due to the requirement for an expensive thermal cycler with a fluorescence detector. among other techniques one promising candidate is a novel nucleic acid amplification method termed loop-mediated isothermal amplification. several investigators have developed lamp assays for detection of pathogenic microorganisims. lamp assay is faster and easier to perform than conventional pcr assays, as well as being more specific. furthermore, because the lamp assay synthesizes a large amount of dna, the products can be detected by simple turbidity. thus, compared to pcr assays, expensive equipment is not necessary to give a high level of precision. these features allow simple, rapid and cost-effective detection. also, the increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of dna synthesized. in addition, the preparation steps of the lamp assay are fewer than with conventional pcr and real-time pcr assays, and lamp assays require less time than those assays. although various lamp assays for the identification of pathogenic organisms have been developed, no assay for the detection of v. parahaemolyticus has been described. here, we describe a sensitive, rapid and simple lamp assay for the detection of v. parahaemolyticus. sensitivity was determined in pure cultures and in spiked shrimp samples.  lamp products were detected from all v. parahaemolyticus strains. no lamp products were detected from any of the non-parahaemolyticus vibrio and non-vibrio strains. the pcr assay required more than h, while the lamp assay was markedly faster, requiring for amplification 13– min in a single colony on tcbs agar from each of v. parahaemolyticus strains and less than min in spiked shrimp samples. the assay required less than min and min for detection of v. parahaemolyticus in a colony on tcbs agar and in spiked shrimp samples from the beginning of dna extraction to final determination. a) described in the methods section. as shown in table sensitivities of the lamp assay for v. parahaemolyticus aq in pure cultures and in spiked shrimp samples were found to be × cfu per ml. further, the sensitivity of the lamp assay was 10-fold higher than that of the pcr assay. the dilutions yielding - cfu per reaction showed an increase in turbidity and was visible as white turbidity but not that of cfu per reaction. sensitivities determined by the two methods were constantly matched with each other. when lamp assay performed in triplicate using μl templates dna containing cfu per reaction from pure cultures, two of three samples showed positive results, as well as using μl templates dna from shrimp samples. +, triplicate assay showed all positive. ±, triplicate assay showed both positive and negative. -, triplicate assay showed all negative. nd, not determined. discussion the bacterial culture test for the isolation and identification of v. parahaemolyticus from food samples after enrichment requires 2– d, with plating onto selective agars, sequential subculture and biochemical characteristic test. in contrast, the lamp assay was markedly faster. conventional pcr assay requires 4– h for amplification, electrophoresis and staining, while the lamp assay requires for dna extraction from samples and amplification less than 40– min. further, amplification of the lamp assay could be judged by visual assessment using the naked eye, without the need for electrophoresis. the lamp assay was more sensitive, rapid and simple than the conventional pcr assay. therefore, the lamp assay is more effective in detecting v. parahaemolyticus than the conventional pcr assay. in the preliminary tests, the lamp assay using and μl templates dna from shrimp samples was enough for amplification without any inhibition of the reaction in μl reaction mixture volumes. the pcr assay was, however, shown inhibited results in μl reaction mixture volumes. no pcr products were detected from the pcr reaction mixtures containing μl templates from shrimp samples. slight bands were detected from the mixtures containing μl templates. clear bands were obtained from each of the mixture containing μl or μl templates. it is known that some components of food inhibit dna amplification such as lamp and pcr assays. the results seem to suggest lamp is more resistant than pcr assay to inhibition by shrimp components. contamination of v. parahaemolyticus in seafood is one of the most important public health hazards. the frequent outbreaks caused by v. parahaemolyticus worldwide highlight the need for control of contamination of v. parahaemolyticus in seafood. especially, ingestion of fresh raw seafood as sushi and sashimi dishes in japan is a risk factor to humans. the total number of v. parahaemolyticus cells in fish fillets and shellfishes is required below cfu/g for raw consumption by law in japan. to reduce the risk of v. parahaemolyticus infection and to ensure food safety, our lamp assay would be significant in the detection of total v. parahaemolyticus in routine tests and in potentially applying to the most-provable-number method. rapid, simple and sensitive assay for detection of v. parahaemolyticus by lamp should facilitate the surveillance for control of contamination of v. parahaemolyticus in seafood. the lamp assay has potential value for rapid and simple screening of total v. parahaemolyticus-contaminated samples before they are consumed. although the shrimp samples were artificially spiked, we successfully developed the first lamp assay for detection of total v. parahaemolyticus from seafood samples. development of the lamp assay for detection of virulent strains of v. parahaemolyticus is required, as well as real-time and conventional pcr assays. further studies will be performed for detection of virulent v. parahaemolyticus and evaluation of the assay using a large number of seafood naturally contaminated by v. parahaemolyticus.  the lamp assay provided markedly more sensitive, simple and rapid detection of v. parahaemolyticus than conventional biochemical and pcr assays. the lamp assay for detection of v. parahaemolyticus required less than min in a colony on tcbs agar and min in spiked shrimp samples from the beginning of dna extraction to final determination. the lamp assay is a powerful tool for the rapid and sensitive detection of v. parahaemolyticus, and will facilitate the surveillance for control of contamination of v. parahaemolyticus in seafood. 
thermostable direct hemolysin and tdh-related hemolysin are known as major virulence determinants of v. parahaemolyticus. most v. parahaemolyticus isolates from the environment do not produce tdh or trh. total v. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. detection of total v. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. the assay provided markedly more sensitive and rapid detection of v. parahaemolyticus strains than conventional biochemical and pcr assays. the assay correctly identified v. parahaemolyticus strains, but did not detect non-parahaemolyticus vibrio and non-vibrio strains. sensitivity of the lamp assay for direct detection of v. parahaemolyticus in pure cultures and in spiked shrimp samples was × cfu per ml/g. the lamp assay was markedly faster, requiring for amplification 13– min in a single colony on tcbs agar from each of v. parahaemolyticus strains and less than min in spiked shrimp samples. the lamp assay for detection of v. parahaemolyticus required less than min in a single colony on thiosulfate citrate bile salt sucrose agar and min in spiked shrimp samples from the beginning of dna extraction to final determination. the lamp assay is a sensitive, rapid and simple tool for the detection of v. parahaemolyticus and will facilitate the surveillance for control of contamination of v. parahaemolyticus in seafood. the sensitivity of the lamp assay was 10-fold more sensitive than that of the conventional pcr assay. vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. thus, we developed a novel and highly specific loop-mediated isothermal amplification assay for the sensitive and rapid detection of vibrio parahaemolyticus.
 vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. thermostable direct hemolysin and tdh-related hemolysin are known as major virulence determinants of v. parahaemolyticus. most v. parahaemolyticus isolates from the environment do not produce tdh or trh. total v. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. detection of total v. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. thus, we developed a novel and highly specific loop-mediated isothermal amplification assay for the sensitive and rapid detection of vibrio parahaemolyticus.  the assay provided markedly more sensitive and rapid detection of v. parahaemolyticus strains than conventional biochemical and pcr assays. the assay correctly identified v. parahaemolyticus strains, but did not detect non-parahaemolyticus vibrio and non-vibrio strains. sensitivity of the lamp assay for direct detection of v. parahaemolyticus in pure cultures and in spiked shrimp samples was × cfu per ml/g. the sensitivity of the lamp assay was 10-fold more sensitive than that of the conventional pcr assay. the lamp assay was markedly faster, requiring for amplification 13– min in a single colony on tcbs agar from each of v. parahaemolyticus strains and less than min in spiked shrimp samples. the lamp assay for detection of v. parahaemolyticus required less than min in a single colony on thiosulfate citrate bile salt sucrose agar and min in spiked shrimp samples from the beginning of dna extraction to final determination.  the lamp assay is a sensitive, rapid and simple tool for the detection of v. parahaemolyticus and will facilitate the surveillance for control of contamination of v. parahaemolyticus in seafood. 