副溶血弧菌分子生物学检测方法的建立及其应用
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摘要
副溶血弧菌(Vibrio Parahaemolyticus, Vp)是引发食源性疾病的重要病原菌之一调查数据表明,50%～70%因食用海产品引发的腹泻病例是由副溶血弧菌引起的。因此,食品中存在副溶血弧菌对人的健康是一种潜在危险,检查食品中副溶血弧菌数量具有重要的实际意义。由于副溶血弧菌在特殊的生长条件下容易进入不可培养但存活的状态(Viable But Nonculturable, VNC),所以传统的生化鉴定结果不一定可靠,并且是一项费时而繁琐的工作。因此,建立一种快速、简洁、准确、敏感的检测食品中副溶血弧菌的分子生物学方法,并对国内食品中副溶血弧菌的污染情况进行调查是一个重要的研究课题。
本文探索并建立了定性检测副溶血弧菌的常规PCR方法和利用地高辛标记的寡聚核酸探针方法；同时,为了达到定量检测副溶血弧菌的目的,我们设计并建立了基于TaqMan探针的单一Real-time PCR方法,并在此基础上建立了双重Real-time PCR技术同步定量检测副溶血弧菌和金黄色葡萄球菌的新方法,并对几种方法进行比较研究；最后利用建立的地高辛标记的寡聚核酸探针方法和Real-time PCR方法对华东地区海产品中副溶血弧菌的污染情况进行调查分析。
试验Ⅰ常规PCR方法检测副溶血弧菌
本试验基于国内外的文献并结合检验检疫局自身检验实际,根据副溶血弧菌的tdh基因设计合成了特异的寡聚核苷酸引物,建立了一种快速检测食品中副溶血弧菌的常规PCR方法,通过对人工布菌样品及检验样品进行试验,结果表明设计的引物具有较强的特异性,扩增产物的DNA片段长度与预期设计相符。本方法可做为一种快速手段,有效地对食品中的上述致病性弧菌进行检测。
试验Ⅱ应用地高辛标记的寡聚核酸探针检测副溶血弧菌
本研究在PCR技术基础上,扩增tdh基因中长为648bp的特异性片段,应用DIG标记试剂盒对特异扩增产物进行标记,标记探针可与副溶血弧菌DNA的tdh基因特异性地杂交,建立了副溶血弧菌的斑点杂交检测方法。特异性试验结果表明,5株副溶血弧菌均能产生杂交斑点,而其他2株非副溶血弧菌均不能杂交斑点。本试验建立的方法可快速检测海产品中的副溶血弧菌。
试验Ⅲ基于TaqMan探针的Real-time PCR方法定量检测副溶血弧菌
根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针,建立了基于TaqMan探针的Real-time PCR。通过对9种细菌(12株菌株)的DNA进行扩增,结果所有4株副溶血弧菌均可产生扩增曲线,其他8株非副溶血弧菌则不产生,证明了引物和探针具有很高的特异性。细菌纯培养和人工布菌的检测敏感度分别为1CFU/PCR反应体系和10CFU/PCR反应体系,相关系数均为0.99(r2=0.99)。整个试验可于1h内完成。该试验建立的方法可快速定量检测海产品中的副溶血弧菌。
试验Ⅳ双重实时定量PCR方法同步检测副溶血弧菌及金黄色葡萄球菌
实验中建立了同步检测副溶血弧菌及金黄色葡萄球菌双重实时定量PCR方法,通过对10株副溶血弧菌及9株金黄色葡萄球菌和其他种类细菌的检测,证明该方法具有很高的特异性,检测灵敏度均小于10CFU/PCR反应。将10倍梯度稀释至终浓度1.0×103CFU/m1到1.0×107CFU/ml纯培养副溶血弧菌和金黄色葡萄球菌的进行定量扩增。循环阈值(Ct值)和初始细胞数的对数相关性好,相关系数为1.0。干扰实验表明DNA混合液的定量检测结果与副溶血弧菌和金黄色葡萄球菌的纯基因组DNA检测结果一致,对检测的特异性和灵敏性无特别的影响。
试验V副溶血弧菌不同检测方法的对比研究
为了比较3种检测方法对海产品中副溶血弧菌检测结果的影响,并探讨这些检测手段的利弊。我们以常规培养鉴定方法作为对照,利用建立的常规PCR方法,寡聚核酸探针方法以及Real-time PCR方法对南京地区采集的150份海产品进行副溶血弧菌检测。结果显示,3种方法的阳性检出率一致,但检测所耗费的时间和成本有所差别。通过对3种方法检测结果的统计分析和比较发现,常规PCR方法适于少量样本的定性检测；寡聚核酸探针技术适于大量样品的筛选；而Real-time PCR则适于对样品的定量检测,但成本较高；在对大量样品进行定量监测时,将寡聚核酸探针技术和Real-time PCR方法结合使用,可节约大量的时间和成本。
试验Ⅵ2005～2006年华东沿海地区海产品中副溶血弧菌污染状况的调查分析
国家食源性疾病监测网发现,我国近年来副溶血性弧菌中毒呈显著上升趋势。为进一步了解华东沿海地区海产品中副溶血性弧菌(Vp)的污染情况,2005年1月～2006年12月对我国华东沿海地区海产品进行监测,共采集882份生的海产品,其中甲壳类340份、虾类284份、鱼类258份,及150份水样。采用实验室建立的寡聚核酸探针技术和Real-time PCR方法进行副溶血性弧菌进行分析。结果显示,33.1%的海产品检测出副溶血弧菌,甲壳类、虾类和鱼类试样中副溶血弧菌阳性率分别为44.1%、20.4%和32.6%；甲壳类、虾类和鱼类阳性试样的Real-time PCR平均定量结果依次为1.0×105/g、7.0×104/g和5.0×104/g。环境水样的副溶血弧菌检出率为11.3%。通过对副溶血弧菌的季节分布情况进行调查和统计分析,发现海产品中副溶血弧菌携带率及其数量以夏秋季节较高,冬春季较少。这些结果证实了副溶血弧菌的消长与季节密切相关,这与副溶血弧菌性食物中毒流行规律也是相一致的。监测结果表明,华沿海地区海产品中副溶血弧菌的污染率较高,必须持续地进行食品中副溶血弧菌的主动监测和污染控制。
Virio parahaemolyticus is primarily involved in causing gastrointestinal illnesses. V. parahaemolyticus is considered to be the causative agent in50%to70%of all cases of diarrhea associated with the consumption of fishery products. Once introduced into food-processing plants, bacteria can persist for a long time. So to dectect V. parahaemolyticus is of great significance. The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. Futhermore, V. parahaemolyticus can enter a viable but nonculturable (VNC) state. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the rapid and sensitive detection of V. parahaemolyticus is necessary. Nucleic acid-based tests have emerged as a useful alternative to traditional testing for the maintenance of a safe seafood supply. In the present study, we have developed a series of nucleic acid based detection methods, including a traditional PCR and a digoxigenin (DIG)-labelled probe method, for the screening of seafood for V. parahaemolyticus. As well, a new technique based on Real-time PCR for quantitative detection V. parahaemolyticus and a multiplex real-time PCR for simultaneous and quantitative detection of V. parahaemolyticus also were established. We then carried out a screening of seafood in Eastern China for the presence of V. parahaemolyticus was carried out base on the mutiplex Real-time PCR and culture method.
1Detetection of Vibrio parahaemolyticus in food by traditional PCR
The purpose of this study is to establish a new method for detection V. parahaemolyticus faster and validate its practicability. The new technique involves designing a primer pair targeting tdh gene and using the primer pair for PCR amplification. The method was tried in4V. parahaemolyticus strains and9non-V. parahaemolyticus strains, and also artifically contaminated food samples. The results showed that the technique offered excellent differentiation and lower detection limit (10CFU/g). The full course of assay could be completed in13hours, which was significantly shorter than the time needed by conventional techniques. It is concluded that the technique is practical with advantages of simple operation, higher specificity, less time consuming and lower detection limit.
2A digoxigenin (DIG)-labelled probe for detection of V. parahaemolyticus
A digoxigenin (DIG)-labelled probe targeted to the tdh gene fragment was prepared by PCR and evaluated for specificity against5strains of V. parahaemolyticus,2strains of non-vibrio species. Of the7isolates tested, the probe hybridized only with the5strains of V. parahaemolyticus, indicating species specificity. The results suggest that the probe is useful for detection of the V. parahaemolyticus tdh gene.
3Application of Real-time PCR for quantitative detection of V. parahaemolyticus
It is important to develop a nucleic acid-based test for quantitative detection of V. parahaemolyticus. A TaqMan PCR assay was presented for quantitative detection of V. parahaemolyticus in pure cultures and oysters. The primers and probe were designed according to the gyrase B gene (gyrB) sequence of V. parahaemolyticus strains on GENEBANK website. Amplification of DNAs from12bacterial strains comprising9genera showed that all of the strains of V. parahaemolyticus tested (n=4) were positive and all other species of strains tested (n=8) were negative.The results of the TaqMan PCR with raw oysters inoculated with V. parahaemolyticus were comparable to those of pure cultures. The sensitivity of the assay was1CFU PCR Mixture-1in pure culture and10CFU PCR mixture-1in inoculated raw oyster, the correlation rate was0.99(r2=0.99). The assay could be completed within1h. The TaqMan probe and primers set developed in this study can be used as a rapid screening tool for the presence of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.
4Multiplex Real-Time PCR for quantitative detection of V. parahaemolyticus and Staphylococcus aureus
A multiplex Real-time PCR assay was developed for the simultaneous and quantitative detection of V. parahaemolyticus and Vibrio vuLnificus in a single tube. The assay was tested on V. parahaemolyticus (n=10),S. aureus (n=9) and other bacteria species, the result showed that the assay was highly specific for V. parahaemolyticus and S. aureus tested. The sensitivity of the assay was demonstrated to less than10copies of bacteria genome DNA of V. parahaemolyticus and S. aureus, respectively. Quantitative linearity of multiplex Real-time PCR was achieved by amplified ten-fold dilutions of purified genomic DNA of V. parahaemolyticus and S. aureus ranging from107to103CFU mL-1based on plate counts, respectively, the log cell number of bacteria and the amount of production (presented by Ct) showed excellent correlation (r2=1.00). Inhibition studies for the multiplex Real-time PCR assay, performed by spiking the DNA extracts from11negative bacteria with purified DNA from V. parahaemolyticus and S. aureus, these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. These data also indicate that multiplex Real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus and S. aureus in seafood.
5Comparative study on detecton methods for V. parahaemolyticus
A comparative study was conducted to determine the efficiency, time consuming and cost of different molecular detction methods for detection for V. parahaemolytiucs from seafood.150samples of seafood, purchased from several local markets in harvest season in Nanjing, were subjected to traditional culture methods, conventional PCR, digoxigenin (DIG)-labelled probe and Real-time PCR assays, respectively. The result was consistent among three molecular methods. The positive ratio for the V. parahaemolyticus detected by three molecular methods was slightly higher than traditional culture methods. However, time consuming and cost is different. The data showed that conventional PCR is more fit for qualitive detection of V. parahaemolyticus from seafood in small number than digoxigenin (DIG)-labelled probe. Digoxigenin (DIG)-labelled probe is suitable for large scale screening of V. parahaemolyticus from seafood. The Real-time PCR can be used as a quantitative tool for the dectiom of V. parahaemolyticus in seafood. The study aslo implicated that the combination of Digoxigenin (DIG)-labelled probe methods and Real-time PCR assay was efficent for large scalely quantitative dection of V. parahaemolyticus.
6Study on the distribution of V. parahaemolyticuand in the seafood in Eastern China
According to the data collected by the National Fodbome Diseases Surveillance Network, the food poisoning caused by V. parahaemolyticus is going up in China recent years. In order to get more information about the V. parahaemolyticus contamination,882samples, including sea shellfish (n=340), shrimp (n=284) and fish (n=258) were collected from several local markets in harvest season in Eastern China within the period from January2005to December2006. The V. parahaemolyticus in seafood samples was determined qualitatively and quantitatively by the digoxigenin (DIG)-labelled probe and Real-time PCR technique. V. parahaemolyticus was isolated from33.1%of all the samples (292/882). High frequency of V. parahaemolyticus was detected in seafoods. Incidence of V. praahaemolyticus in shellfish, shrimp and fish were44.1%,20.4%and29.6%, respectively, with the mean densities of V. parahaemolyticus in positive samples1.0×105/g,7.0×104/g and5.0×104/g, respectively. V. parahaemolyticus was isolated from11.3%of all the water samples (17/150). In addition, the result revealed that the ebb and flow of V. parahaemolyticus related closely to the season. This was consistent to the phenomenon of food poision causing by V. parahaemolyticus. It is concluded that this organism needs to be intensively monitored and controled in raw seafoods.
本文探索并建立了定性检测副溶血弧菌的常规PCR方法和利用地高辛标记的寡聚核酸探针方法；同时,为了达到定量检测副溶血弧菌的目的,我们设计并建立了基于TaqMan探针的单一Real-time PCR方法,并在此基础上建立了双重Real-time PCR技术同步定量检测副溶血弧菌和金黄色葡萄球菌的新方法,并对几种方法进行比较研究；最后利用建立的地高辛标记的寡聚核酸探针方法和Real-time PCR方法对华东地区海产品中副溶血弧菌的污染情况进行调查分析。
试验Ⅰ常规PCR方法检测副溶血弧菌
本试验基于国内外的文献并结合检验检疫局自身检验实际,根据副溶血弧菌的tdh基因设计合成了特异的寡聚核苷酸引物,建立了一种快速检测食品中副溶血弧菌的常规PCR方法,通过对人工布菌样品及检验样品进行试验,结果表明设计的引物具有较强的特异性,扩增产物的DNA片段长度与预期设计相符。本方法可做为一种快速手段,有效地对食品中的上述致病性弧菌进行检测。
试验Ⅱ应用地高辛标记的寡聚核酸探针检测副溶血弧菌
本研究在PCR技术基础上,扩增tdh基因中长为648bp的特异性片段,应用DIG标记试剂盒对特异扩增产物进行标记,标记探针可与副溶血弧菌DNA的tdh基因特异性地杂交,建立了副溶血弧菌的斑点杂交检测方法。特异性试验结果表明,5株副溶血弧菌均能产生杂交斑点,而其他2株非副溶血弧菌均不能杂交斑点。本试验建立的方法可快速检测海产品中的副溶血弧菌。
试验Ⅲ基于TaqMan探针的Real-time PCR方法定量检测副溶血弧菌
根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针,建立了基于TaqMan探针的Real-time PCR。通过对9种细菌(12株菌株)的DNA进行扩增,结果所有4株副溶血弧菌均可产生扩增曲线,其他8株非副溶血弧菌则不产生,证明了引物和探针具有很高的特异性。细菌纯培养和人工布菌的检测敏感度分别为1CFU/PCR反应体系和10CFU/PCR反应体系,相关系数均为0.99(r2=0.99)。整个试验可于1h内完成。该试验建立的方法可快速定量检测海产品中的副溶血弧菌。
试验Ⅳ双重实时定量PCR方法同步检测副溶血弧菌及金黄色葡萄球菌
实验中建立了同步检测副溶血弧菌及金黄色葡萄球菌双重实时定量PCR方法,通过对10株副溶血弧菌及9株金黄色葡萄球菌和其他种类细菌的检测,证明该方法具有很高的特异性,检测灵敏度均小于10CFU/PCR反应。将10倍梯度稀释至终浓度1.0×103CFU/m1到1.0×107CFU/ml纯培养副溶血弧菌和金黄色葡萄球菌的进行定量扩增。循环阈值(Ct值)和初始细胞数的对数相关性好,相关系数为1.0。干扰实验表明DNA混合液的定量检测结果与副溶血弧菌和金黄色葡萄球菌的纯基因组DNA检测结果一致,对检测的特异性和灵敏性无特别的影响。
试验V副溶血弧菌不同检测方法的对比研究
为了比较3种检测方法对海产品中副溶血弧菌检测结果的影响,并探讨这些检测手段的利弊。我们以常规培养鉴定方法作为对照,利用建立的常规PCR方法,寡聚核酸探针方法以及Real-time PCR方法对南京地区采集的150份海产品进行副溶血弧菌检测。结果显示,3种方法的阳性检出率一致,但检测所耗费的时间和成本有所差别。通过对3种方法检测结果的统计分析和比较发现,常规PCR方法适于少量样本的定性检测；寡聚核酸探针技术适于大量样品的筛选；而Real-time PCR则适于对样品的定量检测,但成本较高；在对大量样品进行定量监测时,将寡聚核酸探针技术和Real-time PCR方法结合使用,可节约大量的时间和成本。
试验Ⅵ2005～2006年华东沿海地区海产品中副溶血弧菌污染状况的调查分析
国家食源性疾病监测网发现,我国近年来副溶血性弧菌中毒呈显著上升趋势。为进一步了解华东沿海地区海产品中副溶血性弧菌(Vp)的污染情况,2005年1月～2006年12月对我国华东沿海地区海产品进行监测,共采集882份生的海产品,其中甲壳类340份、虾类284份、鱼类258份,及150份水样。采用实验室建立的寡聚核酸探针技术和Real-time PCR方法进行副溶血性弧菌进行分析。结果显示,33.1%的海产品检测出副溶血弧菌,甲壳类、虾类和鱼类试样中副溶血弧菌阳性率分别为44.1%、20.4%和32.6%；甲壳类、虾类和鱼类阳性试样的Real-time PCR平均定量结果依次为1.0×105/g、7.0×104/g和5.0×104/g。环境水样的副溶血弧菌检出率为11.3%。通过对副溶血弧菌的季节分布情况进行调查和统计分析,发现海产品中副溶血弧菌携带率及其数量以夏秋季节较高,冬春季较少。这些结果证实了副溶血弧菌的消长与季节密切相关,这与副溶血弧菌性食物中毒流行规律也是相一致的。监测结果表明,华沿海地区海产品中副溶血弧菌的污染率较高,必须持续地进行食品中副溶血弧菌的主动监测和污染控制。
Virio parahaemolyticus is primarily involved in causing gastrointestinal illnesses. V. parahaemolyticus is considered to be the causative agent in50%to70%of all cases of diarrhea associated with the consumption of fishery products. Once introduced into food-processing plants, bacteria can persist for a long time. So to dectect V. parahaemolyticus is of great significance. The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. Futhermore, V. parahaemolyticus can enter a viable but nonculturable (VNC) state. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the rapid and sensitive detection of V. parahaemolyticus is necessary. Nucleic acid-based tests have emerged as a useful alternative to traditional testing for the maintenance of a safe seafood supply. In the present study, we have developed a series of nucleic acid based detection methods, including a traditional PCR and a digoxigenin (DIG)-labelled probe method, for the screening of seafood for V. parahaemolyticus. As well, a new technique based on Real-time PCR for quantitative detection V. parahaemolyticus and a multiplex real-time PCR for simultaneous and quantitative detection of V. parahaemolyticus also were established. We then carried out a screening of seafood in Eastern China for the presence of V. parahaemolyticus was carried out base on the mutiplex Real-time PCR and culture method.
1Detetection of Vibrio parahaemolyticus in food by traditional PCR
The purpose of this study is to establish a new method for detection V. parahaemolyticus faster and validate its practicability. The new technique involves designing a primer pair targeting tdh gene and using the primer pair for PCR amplification. The method was tried in4V. parahaemolyticus strains and9non-V. parahaemolyticus strains, and also artifically contaminated food samples. The results showed that the technique offered excellent differentiation and lower detection limit (10CFU/g). The full course of assay could be completed in13hours, which was significantly shorter than the time needed by conventional techniques. It is concluded that the technique is practical with advantages of simple operation, higher specificity, less time consuming and lower detection limit.
2A digoxigenin (DIG)-labelled probe for detection of V. parahaemolyticus
A digoxigenin (DIG)-labelled probe targeted to the tdh gene fragment was prepared by PCR and evaluated for specificity against5strains of V. parahaemolyticus,2strains of non-vibrio species. Of the7isolates tested, the probe hybridized only with the5strains of V. parahaemolyticus, indicating species specificity. The results suggest that the probe is useful for detection of the V. parahaemolyticus tdh gene.
3Application of Real-time PCR for quantitative detection of V. parahaemolyticus
It is important to develop a nucleic acid-based test for quantitative detection of V. parahaemolyticus. A TaqMan PCR assay was presented for quantitative detection of V. parahaemolyticus in pure cultures and oysters. The primers and probe were designed according to the gyrase B gene (gyrB) sequence of V. parahaemolyticus strains on GENEBANK website. Amplification of DNAs from12bacterial strains comprising9genera showed that all of the strains of V. parahaemolyticus tested (n=4) were positive and all other species of strains tested (n=8) were negative.The results of the TaqMan PCR with raw oysters inoculated with V. parahaemolyticus were comparable to those of pure cultures. The sensitivity of the assay was1CFU PCR Mixture-1in pure culture and10CFU PCR mixture-1in inoculated raw oyster, the correlation rate was0.99(r2=0.99). The assay could be completed within1h. The TaqMan probe and primers set developed in this study can be used as a rapid screening tool for the presence of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.
4Multiplex Real-Time PCR for quantitative detection of V. parahaemolyticus and Staphylococcus aureus
A multiplex Real-time PCR assay was developed for the simultaneous and quantitative detection of V. parahaemolyticus and Vibrio vuLnificus in a single tube. The assay was tested on V. parahaemolyticus (n=10),S. aureus (n=9) and other bacteria species, the result showed that the assay was highly specific for V. parahaemolyticus and S. aureus tested. The sensitivity of the assay was demonstrated to less than10copies of bacteria genome DNA of V. parahaemolyticus and S. aureus, respectively. Quantitative linearity of multiplex Real-time PCR was achieved by amplified ten-fold dilutions of purified genomic DNA of V. parahaemolyticus and S. aureus ranging from107to103CFU mL-1based on plate counts, respectively, the log cell number of bacteria and the amount of production (presented by Ct) showed excellent correlation (r2=1.00). Inhibition studies for the multiplex Real-time PCR assay, performed by spiking the DNA extracts from11negative bacteria with purified DNA from V. parahaemolyticus and S. aureus, these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. These data also indicate that multiplex Real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus and S. aureus in seafood.
5Comparative study on detecton methods for V. parahaemolyticus
A comparative study was conducted to determine the efficiency, time consuming and cost of different molecular detction methods for detection for V. parahaemolytiucs from seafood.150samples of seafood, purchased from several local markets in harvest season in Nanjing, were subjected to traditional culture methods, conventional PCR, digoxigenin (DIG)-labelled probe and Real-time PCR assays, respectively. The result was consistent among three molecular methods. The positive ratio for the V. parahaemolyticus detected by three molecular methods was slightly higher than traditional culture methods. However, time consuming and cost is different. The data showed that conventional PCR is more fit for qualitive detection of V. parahaemolyticus from seafood in small number than digoxigenin (DIG)-labelled probe. Digoxigenin (DIG)-labelled probe is suitable for large scale screening of V. parahaemolyticus from seafood. The Real-time PCR can be used as a quantitative tool for the dectiom of V. parahaemolyticus in seafood. The study aslo implicated that the combination of Digoxigenin (DIG)-labelled probe methods and Real-time PCR assay was efficent for large scalely quantitative dection of V. parahaemolyticus.
6Study on the distribution of V. parahaemolyticuand in the seafood in Eastern China
According to the data collected by the National Fodbome Diseases Surveillance Network, the food poisoning caused by V. parahaemolyticus is going up in China recent years. In order to get more information about the V. parahaemolyticus contamination,882samples, including sea shellfish (n=340), shrimp (n=284) and fish (n=258) were collected from several local markets in harvest season in Eastern China within the period from January2005to December2006. The V. parahaemolyticus in seafood samples was determined qualitatively and quantitatively by the digoxigenin (DIG)-labelled probe and Real-time PCR technique. V. parahaemolyticus was isolated from33.1%of all the samples (292/882). High frequency of V. parahaemolyticus was detected in seafoods. Incidence of V. praahaemolyticus in shellfish, shrimp and fish were44.1%,20.4%and29.6%, respectively, with the mean densities of V. parahaemolyticus in positive samples1.0×105/g,7.0×104/g and5.0×104/g, respectively. V. parahaemolyticus was isolated from11.3%of all the water samples (17/150). In addition, the result revealed that the ebb and flow of V. parahaemolyticus related closely to the season. This was consistent to the phenomenon of food poision causing by V. parahaemolyticus. It is concluded that this organism needs to be intensively monitored and controled in raw seafoods.
引文
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