副溶血弧菌高通量分子检测方法的建立与基因分型研究
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摘要
副溶血弧菌(Vibrio parahaemolyticus,VP)是一种革兰氏阴性嗜盐杆菌,主要存在于近岸海水、海底沉积物和鱼、虾以及贝类等海洋生物中,人类食用了含有该菌的生的或者未煮熟的水产品能引起食物中毒、反应性关节炎和心脏疾病。此外,它还严重危害了水产养殖业的发展。因此,了解副溶血弧菌在水产品中的分布特征以及建立快速、灵敏的检测方法对于食品安全监控具有重要意义。
本课题主要对副溶血弧菌在水产品中的分布特征、基因分型以及快速检测技术进行了比较深入的研究,主要研究内容和结果如下:
1.副溶血弧菌多重PCR检测方法的建立及评价
利用本地BLAST序列比对分析方法挖掘到副溶血弧菌新的种特异性基因,即D型氨基酸脱氢酶小亚基基因(D-amino acid dehydrogenase, small subunit, aadS),根据该基因设计用于PCR检测的特异性引物,并对引物的特异性和灵敏度进行了系统的生物学评价,证实该基因特异性强,灵敏度高,能够作为副溶血弧菌分子检测的靶点。另外,结合毒力因子耐热直接溶血素(tdh)和tdh相关溶血素基因(trh),建立了副溶血弧菌多重PCR检测方法,并对该方法进行了系统评价。该多重PCR方法特异性为100%,基因组DNA检测灵敏度为20 fg/PCR。将该方法应用于55份实际样品(虾,鱼和牡蛎等)的检测,通过与传统国标方法进行比较,发现有一份样品用国标检测为阳性,而该多重PCR方法检测为阴性,其它样品检测结果都与传统方法一致。表明本课题建立的副溶血弧菌多重PCR检测方法具有快速、特异性强和灵敏度高等优点,可用于水产品和临床样本的快速检测,同时可对分离菌株是否具有致病性能够进行初步判断。
2.基于单碱基延伸标签法的基因芯片检测方法的建立
在多重PCR基础上建立了基于单碱基延伸标签法(SBE-TAGS)的基因芯片检测方法,并可用于水产品中副溶血弧菌、霍乱弧菌、创伤弧菌、溶藻弧菌、拟态弧菌、哈维氏弧菌和鳗弧菌的同时检测。利用三组多重PCR实验同时扩增7种水产致病弧菌9个特异性检测靶基因。根据aadS, tdh和trh基因的检测结果来判断水产品中是否含有副溶血弧菌及其是否具有致病性;依据col, toxR和vvh基因分别检测溶藻弧菌、拟态弧菌和创伤弧菌;采用empA, vhh1和tcpA基因依次检测鳗弧菌、哈维氏弧菌和霍乱弧菌。纯化后的多重PCR产物进行单碱基延伸反应后被标记上荧光染料Cy3,标记后的产物进行芯片杂交、信号扫描和结果分析。结果表明,该基因芯片检测特异性为100%,检测灵敏度为200 fg/PCR～20 pg/PCR。将该方法应用于55份实际水产样品的检测,发现有18份样品同时含有副溶血弧菌和溶藻弧菌;通过与传统国标方法进行比较,发现除一份样品与传统方法检测结果不同外,其它样品检测结果均为一致。该方法的建立不但可以保障为消费者提供更加安全的水产品,还有利于水产养殖业的健康发展。
3.副溶血弧菌在水产品中分布特征及多样性分析
利用国家标准方法和PCR方法相结合的手段,在2006年4月至2009年6月之间,从上海地区水产品批发市场、零售市场以及大型超市合计取样368份。从上海地区215份样品中分离到47株副溶血弧菌,分离率为21%;从进口的153份样品中分离到56株副溶血弧菌,分离率为37%。对分离菌株进行毒力基因PCR检测和尿酶实验测定,发现上海地区副溶血弧菌分离菌株中有2株(2/47,4%)含有tdh基因但不含trh基因,且尿酶实验均为阴性。进口样品中副溶血弧菌分离菌株有4株(4/56,7%)携带tdh基因,其中有一株菌株(1/56,2%)既含有tdh基因,又含有trh基因,尿酶实验也为阳性,其余菌株尿酶均为阴性。经O抗原测定,发现上海地区副溶血弧菌分离菌株O抗原主要为O1和O2,其中O1有14株(14/47,30%),O2有10株(10/47,22%)。进口样品中副溶血弧菌分离株O抗原主要为O10和O2,其中O10有13株(13/56,23%),O2有10株(10/56,18%)。为了了解不同地区分离菌株之间的相关性,将103株副溶血弧菌食品分离菌株和上海地区5株常见的不同血清型的副溶血弧菌临床分离菌株同时经过肠杆菌基因间重复序列PCR(ERIC-PCR)分型,利用数学软件BioNumercis version 5.0根据非加权算术平均数聚类法(UPGMA)对分型结果进行处理和分析,如果按相似性值大于0.70,所有分离菌株被分成44个类群,发现有4株上海地区样品分离株(SJTUF30004, SJTUF30013, SJTUF30025和SJTUF30037)和6株进口样品分离株( SJTUF30083, SJTUF30086, SJTUF30089 ,SJTUF30090,SJTUF30094和SJTUF30102)与临床致病菌株聚在一起,表明这10株分离菌株与临床分离株在基因水平存在较高的相似性,据此推测它们具有潜在的致病可能性。
4.副溶血弧菌基因分型及菌株相关性分析
鉴于副溶血弧菌在我国沿海地区引起的食物中毒事件频频发生,因此迫切需要对该菌传染源的感染地点和流行毒力菌株做出快速正确的鉴定和确认。本研究以上海及周边地区流行病学上不相关的56株分离菌株(2006年至2008年)为研究对象,这些菌株同时经过四种基因分型方法进行分析,包括ERIC-PCR、核糖体分型、脉冲场凝胶电泳(PFGE)和gyrB基因序列分析分型。四种分型方法结果通过数据处理软件Bionumeircus version 5.0根据非加权算术平均数聚类法(UPGMA)进行聚类分析和分子进化树的构建,聚类结果显示,每种分型方法得到的结果并不完全一致,而且差异较大。根据相似性值大于0.76,按辛普森方程计算每种方法的分辨率(D值),ERIC-PCR、PFGE、核糖体分型和gyrB基因的序列分型的分辨率依次为0.942、0.907、0.756和0.702。由于核糖体分型的分辨率较低,说明它不太适合用于同一地区副溶血弧菌分离菌株之间的亚种分型。四种方法聚类分析的结果同时发现,两株食品分离菌株F13和QS2均和致病菌株聚成一类,反映出它们和致病菌株之间的亲缘关系较近,具有潜在的致病可能性。通过对56株副溶血弧菌分离株的基因分型研究,发现ERIC-PCR和gyrB基因序列分析联合起来使用,不但具有较高的分辨率(D=0.966),而且还可以快速比较不同来源的副溶血弧菌分离菌株之间的相关性。
Vibrio parahaemolyticus (VP) is a halophilic gram-negative bacterium disseminated in marine and estuarine environments worldwide,especially in fishes, shellfish and other seafood products. It is one of the most important pathogens causing seafood-borne gastroenteritis associated with the consumption of raw or partially cooked seafood. Additionally, V. parahaemolyticus has shown great harm to the development of the aquaculture. Molecular distribution characteristics of Vibrio parahaemolyticus and rapid detection of this organism are of great importance.
The major contributions of the research were described as follows:
1. Establishment and evaluation of multiplex PCR for the detection of Vibrio parahaemolyticus
One new species-specific target gene (aadS), encoding D-amino acid dehydrogenase, small subunit of Vibrio parahaemolyticus was obtained by bioinformatics analysis, and primers were designed for target gene PCR detection. Because its pathogenicity was strongly correlated to two well-characterized hemolysins, the thermostable direct hemolysin (tdh) and the tdh-related hemolysin (trh), multiplex PCR were established and evaluated based on the target genes aadS, tdhand trh. The detection specificity of this method was 100%, with the sensitivity for pure genomic DNA at 20 fg per reaction. Application of the method was involved in detecting 55 naturally contaminated seafood samples (shrimp, fish, and oysters) compared with conventional assays (microbiological and biochemical tests). It turned out that only one sample was identified negative by the multiplex PCR assay but positive by the conventional method. Therefore, the multiplex PCR method established in this study is accurate, sensitive, specific and applicable for the detection of V. parahaemolyticus. It can be a supplement of conventional method to be applied to seafood detection and clinical analysis to increase detection efficiency and determine pathogenicity of the isolates.
2. Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood
A single base extension-tag array on glass slides (SBE-TAGS) microarray based on the multiplex PCR was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, V. cholerae, V. vulnificus, V. mimicus, V. alginolyticus, V. anguillarum, and V. harveyi. Three multiplex PCR systems were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi and V. cholera respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%, of all samples, there were 18 samples which contained V. parahaemolyticus and V. alginolyticus. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.
3. Distribution characteristics and diversity of V. parahaemolyticus in seafood
There were 368 seafood samples including 215 from Shanghai area and 153 from the other countries, which were taken from the wholesale markets, retail markets and the supermarkets in Shanghai area between April 2006 and June 2009. V. parahaemolyticus was isolated by combining the method of national standard with PCR. There were 47 V. parahaemolyticus strains which were isolated from Shanghi area samples, and the isolation rate was 21%. There were 56 V. parahaemolyticus strains which were isolated from the samples which originly came from the other countries, and isolation rate was 37%. The V. parahaemolyticus isolates from Shanghai area with the hemolysin genes were low, and only two isolates (2/47,4%) carried the the thermostable direct hemolysin (tdh) without the tdh-related hemolysin (trh), and the urease experiment was negative for all of the isolates from Shanghai area. The isolates from the other countries with the hemolysin genes were also low, and there were only four isolates (4/56,7%) with tdh gene and only one isolate from above four isolates carried both tdh gene and trh gene with positive result for the urease experiment. Serotypes of all the isolates were measured by agglutination using a commercial kit of O-Antigen according to the manufacturer’s specifications, fourteen isolates from Shanghai area consisted of O1 (14/47, 30%), ten isolates consisted of O2 (10/47, 22%). On the other hand, thirteen isolates from the other countries consisted of O10 (14/56, 23%), and ten isolates consisted of O2 (10/56, 18%). All 103 isolates from the seafood samples and 5 major clinial isolates were typed by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and there were 44 patterns possessed by applying the cluster analysis of genetic profiles from ERIC-PCR typing to all strains based on the similarity value of more than 70% using BioNumerics version 5.0 by the unweighted-pair group method using average linkages. Genetic profiles of cluster analysis from ERIC-PCR clearly showed that four isolates (SJTUF30004, SJTUF30013, SJTUF30025 and SJTUF30037) from Shanghai area and six isolates (SJTUF30083, SJTUF30086, SJTUF30089 ,SJTUF30090,SJTUF30094 and SJTUF30102) from the other countries that originated from seafood were clustered together which demonstrated that they were potentially pathogenic.
4. Genetyping and the relatedness among the V. parahaemolyticus isolates
The outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlight the need for strain characterization and differentiation of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China between 2006 and 2008. The isolates were characterized using four various molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly by the unweighted-pair group method using average linkages showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity based on the similarity value more than 0.76. The discriminatory index of ERIC–PCR typing was maximal (D﹦0.942), while that of sequence analysis of the gyrB gene was minimal (D﹦0.702), and the discriminatory index of PFGE and ribotyping was 0.907 and 0.756. Therefore, ribotyping exhibited a discriminatory ability much lower than PFGE and ERIC-PCR, and may have limited application in V. parahaemolyticus subtyping and outbreak investigation. In conclusion, our results suggest that ERIC-PCR used in conjunction with gyrB sequence analysis would provide a reliable and accurate typing strategy for V. parahaemolyticus. This combination of techniques exhibits an excellent discrimination (D=0.966) and can be used as a rapid means of comparing V. parahaemolyticus isolates for epidemiological investigations.
本课题主要对副溶血弧菌在水产品中的分布特征、基因分型以及快速检测技术进行了比较深入的研究,主要研究内容和结果如下:
1.副溶血弧菌多重PCR检测方法的建立及评价
利用本地BLAST序列比对分析方法挖掘到副溶血弧菌新的种特异性基因,即D型氨基酸脱氢酶小亚基基因(D-amino acid dehydrogenase, small subunit, aadS),根据该基因设计用于PCR检测的特异性引物,并对引物的特异性和灵敏度进行了系统的生物学评价,证实该基因特异性强,灵敏度高,能够作为副溶血弧菌分子检测的靶点。另外,结合毒力因子耐热直接溶血素(tdh)和tdh相关溶血素基因(trh),建立了副溶血弧菌多重PCR检测方法,并对该方法进行了系统评价。该多重PCR方法特异性为100%,基因组DNA检测灵敏度为20 fg/PCR。将该方法应用于55份实际样品(虾,鱼和牡蛎等)的检测,通过与传统国标方法进行比较,发现有一份样品用国标检测为阳性,而该多重PCR方法检测为阴性,其它样品检测结果都与传统方法一致。表明本课题建立的副溶血弧菌多重PCR检测方法具有快速、特异性强和灵敏度高等优点,可用于水产品和临床样本的快速检测,同时可对分离菌株是否具有致病性能够进行初步判断。
2.基于单碱基延伸标签法的基因芯片检测方法的建立
在多重PCR基础上建立了基于单碱基延伸标签法(SBE-TAGS)的基因芯片检测方法,并可用于水产品中副溶血弧菌、霍乱弧菌、创伤弧菌、溶藻弧菌、拟态弧菌、哈维氏弧菌和鳗弧菌的同时检测。利用三组多重PCR实验同时扩增7种水产致病弧菌9个特异性检测靶基因。根据aadS, tdh和trh基因的检测结果来判断水产品中是否含有副溶血弧菌及其是否具有致病性;依据col, toxR和vvh基因分别检测溶藻弧菌、拟态弧菌和创伤弧菌;采用empA, vhh1和tcpA基因依次检测鳗弧菌、哈维氏弧菌和霍乱弧菌。纯化后的多重PCR产物进行单碱基延伸反应后被标记上荧光染料Cy3,标记后的产物进行芯片杂交、信号扫描和结果分析。结果表明,该基因芯片检测特异性为100%,检测灵敏度为200 fg/PCR～20 pg/PCR。将该方法应用于55份实际水产样品的检测,发现有18份样品同时含有副溶血弧菌和溶藻弧菌;通过与传统国标方法进行比较,发现除一份样品与传统方法检测结果不同外,其它样品检测结果均为一致。该方法的建立不但可以保障为消费者提供更加安全的水产品,还有利于水产养殖业的健康发展。
3.副溶血弧菌在水产品中分布特征及多样性分析
利用国家标准方法和PCR方法相结合的手段,在2006年4月至2009年6月之间,从上海地区水产品批发市场、零售市场以及大型超市合计取样368份。从上海地区215份样品中分离到47株副溶血弧菌,分离率为21%;从进口的153份样品中分离到56株副溶血弧菌,分离率为37%。对分离菌株进行毒力基因PCR检测和尿酶实验测定,发现上海地区副溶血弧菌分离菌株中有2株(2/47,4%)含有tdh基因但不含trh基因,且尿酶实验均为阴性。进口样品中副溶血弧菌分离菌株有4株(4/56,7%)携带tdh基因,其中有一株菌株(1/56,2%)既含有tdh基因,又含有trh基因,尿酶实验也为阳性,其余菌株尿酶均为阴性。经O抗原测定,发现上海地区副溶血弧菌分离菌株O抗原主要为O1和O2,其中O1有14株(14/47,30%),O2有10株(10/47,22%)。进口样品中副溶血弧菌分离株O抗原主要为O10和O2,其中O10有13株(13/56,23%),O2有10株(10/56,18%)。为了了解不同地区分离菌株之间的相关性,将103株副溶血弧菌食品分离菌株和上海地区5株常见的不同血清型的副溶血弧菌临床分离菌株同时经过肠杆菌基因间重复序列PCR(ERIC-PCR)分型,利用数学软件BioNumercis version 5.0根据非加权算术平均数聚类法(UPGMA)对分型结果进行处理和分析,如果按相似性值大于0.70,所有分离菌株被分成44个类群,发现有4株上海地区样品分离株(SJTUF30004, SJTUF30013, SJTUF30025和SJTUF30037)和6株进口样品分离株( SJTUF30083, SJTUF30086, SJTUF30089 ,SJTUF30090,SJTUF30094和SJTUF30102)与临床致病菌株聚在一起,表明这10株分离菌株与临床分离株在基因水平存在较高的相似性,据此推测它们具有潜在的致病可能性。
4.副溶血弧菌基因分型及菌株相关性分析
鉴于副溶血弧菌在我国沿海地区引起的食物中毒事件频频发生,因此迫切需要对该菌传染源的感染地点和流行毒力菌株做出快速正确的鉴定和确认。本研究以上海及周边地区流行病学上不相关的56株分离菌株(2006年至2008年)为研究对象,这些菌株同时经过四种基因分型方法进行分析,包括ERIC-PCR、核糖体分型、脉冲场凝胶电泳(PFGE)和gyrB基因序列分析分型。四种分型方法结果通过数据处理软件Bionumeircus version 5.0根据非加权算术平均数聚类法(UPGMA)进行聚类分析和分子进化树的构建,聚类结果显示,每种分型方法得到的结果并不完全一致,而且差异较大。根据相似性值大于0.76,按辛普森方程计算每种方法的分辨率(D值),ERIC-PCR、PFGE、核糖体分型和gyrB基因的序列分型的分辨率依次为0.942、0.907、0.756和0.702。由于核糖体分型的分辨率较低,说明它不太适合用于同一地区副溶血弧菌分离菌株之间的亚种分型。四种方法聚类分析的结果同时发现,两株食品分离菌株F13和QS2均和致病菌株聚成一类,反映出它们和致病菌株之间的亲缘关系较近,具有潜在的致病可能性。通过对56株副溶血弧菌分离株的基因分型研究,发现ERIC-PCR和gyrB基因序列分析联合起来使用,不但具有较高的分辨率(D=0.966),而且还可以快速比较不同来源的副溶血弧菌分离菌株之间的相关性。
Vibrio parahaemolyticus (VP) is a halophilic gram-negative bacterium disseminated in marine and estuarine environments worldwide,especially in fishes, shellfish and other seafood products. It is one of the most important pathogens causing seafood-borne gastroenteritis associated with the consumption of raw or partially cooked seafood. Additionally, V. parahaemolyticus has shown great harm to the development of the aquaculture. Molecular distribution characteristics of Vibrio parahaemolyticus and rapid detection of this organism are of great importance.
The major contributions of the research were described as follows:
1. Establishment and evaluation of multiplex PCR for the detection of Vibrio parahaemolyticus
One new species-specific target gene (aadS), encoding D-amino acid dehydrogenase, small subunit of Vibrio parahaemolyticus was obtained by bioinformatics analysis, and primers were designed for target gene PCR detection. Because its pathogenicity was strongly correlated to two well-characterized hemolysins, the thermostable direct hemolysin (tdh) and the tdh-related hemolysin (trh), multiplex PCR were established and evaluated based on the target genes aadS, tdhand trh. The detection specificity of this method was 100%, with the sensitivity for pure genomic DNA at 20 fg per reaction. Application of the method was involved in detecting 55 naturally contaminated seafood samples (shrimp, fish, and oysters) compared with conventional assays (microbiological and biochemical tests). It turned out that only one sample was identified negative by the multiplex PCR assay but positive by the conventional method. Therefore, the multiplex PCR method established in this study is accurate, sensitive, specific and applicable for the detection of V. parahaemolyticus. It can be a supplement of conventional method to be applied to seafood detection and clinical analysis to increase detection efficiency and determine pathogenicity of the isolates.
2. Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood
A single base extension-tag array on glass slides (SBE-TAGS) microarray based on the multiplex PCR was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, V. cholerae, V. vulnificus, V. mimicus, V. alginolyticus, V. anguillarum, and V. harveyi. Three multiplex PCR systems were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi and V. cholera respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%, of all samples, there were 18 samples which contained V. parahaemolyticus and V. alginolyticus. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.
3. Distribution characteristics and diversity of V. parahaemolyticus in seafood
There were 368 seafood samples including 215 from Shanghai area and 153 from the other countries, which were taken from the wholesale markets, retail markets and the supermarkets in Shanghai area between April 2006 and June 2009. V. parahaemolyticus was isolated by combining the method of national standard with PCR. There were 47 V. parahaemolyticus strains which were isolated from Shanghi area samples, and the isolation rate was 21%. There were 56 V. parahaemolyticus strains which were isolated from the samples which originly came from the other countries, and isolation rate was 37%. The V. parahaemolyticus isolates from Shanghai area with the hemolysin genes were low, and only two isolates (2/47,4%) carried the the thermostable direct hemolysin (tdh) without the tdh-related hemolysin (trh), and the urease experiment was negative for all of the isolates from Shanghai area. The isolates from the other countries with the hemolysin genes were also low, and there were only four isolates (4/56,7%) with tdh gene and only one isolate from above four isolates carried both tdh gene and trh gene with positive result for the urease experiment. Serotypes of all the isolates were measured by agglutination using a commercial kit of O-Antigen according to the manufacturer’s specifications, fourteen isolates from Shanghai area consisted of O1 (14/47, 30%), ten isolates consisted of O2 (10/47, 22%). On the other hand, thirteen isolates from the other countries consisted of O10 (14/56, 23%), and ten isolates consisted of O2 (10/56, 18%). All 103 isolates from the seafood samples and 5 major clinial isolates were typed by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and there were 44 patterns possessed by applying the cluster analysis of genetic profiles from ERIC-PCR typing to all strains based on the similarity value of more than 70% using BioNumerics version 5.0 by the unweighted-pair group method using average linkages. Genetic profiles of cluster analysis from ERIC-PCR clearly showed that four isolates (SJTUF30004, SJTUF30013, SJTUF30025 and SJTUF30037) from Shanghai area and six isolates (SJTUF30083, SJTUF30086, SJTUF30089 ,SJTUF30090,SJTUF30094 and SJTUF30102) from the other countries that originated from seafood were clustered together which demonstrated that they were potentially pathogenic.
4. Genetyping and the relatedness among the V. parahaemolyticus isolates
The outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlight the need for strain characterization and differentiation of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China between 2006 and 2008. The isolates were characterized using four various molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly by the unweighted-pair group method using average linkages showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity based on the similarity value more than 0.76. The discriminatory index of ERIC–PCR typing was maximal (D﹦0.942), while that of sequence analysis of the gyrB gene was minimal (D﹦0.702), and the discriminatory index of PFGE and ribotyping was 0.907 and 0.756. Therefore, ribotyping exhibited a discriminatory ability much lower than PFGE and ERIC-PCR, and may have limited application in V. parahaemolyticus subtyping and outbreak investigation. In conclusion, our results suggest that ERIC-PCR used in conjunction with gyrB sequence analysis would provide a reliable and accurate typing strategy for V. parahaemolyticus. This combination of techniques exhibits an excellent discrimination (D=0.966) and can be used as a rapid means of comparing V. parahaemolyticus isolates for epidemiological investigations.
引文
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