荧光定量PCR法检测副溶血弧菌tlh和tdh基因的表达差异

副溶血弧菌是广泛存在于近海区域,盐湖和海产品中的食源性致病菌,会引起大规模的食物中毒。TLH(不耐热溶血毒素)和TDH(耐热直接溶血毒素)是副溶血弧菌最主要的毒力基因,通过比较毒力基因的表达量可以间接比较同种菌株在不同应激条件下以及不同菌株之间的毒力差异。本文以在不同条件下培养的3株Vp为材料,分别提取其总RNA,以16S rRNA为内标基因,运用荧光定量PCR技术检测副溶血弧菌TLH和TDH基因在不同应激条件下的表达差异。结果表明:不同菌株和同种菌株在不同应激条件下tlh、tdh基因表达差异均显著;tlh的最适表达条件分别为5%盐度和20°C;tdh的最适表达条件分别为1%盐度和25°C。运用SPSS软件对实验结果进行统计学分析表明:菌株对tlh表达的影响大于盐度大于温度;菌株对tdh表达的影响大于温度大于盐度。     

Vibrio parahaemolyticus serovar O3:K6 gastroenteritis in northeast Brazil

Aims:  To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil.
Methods and Results:  Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl , tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S–23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S–23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh ^– Kanagawa-negative isolates.
Conclusions:  V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless.
Significance and Impact of the Study:  Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this micro-organism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.     

Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment

AIMS: To study the incidence of Vibrio parahaemolyticus in seafoods, water and sediment by molecular techniques vs conventional microbiological methods. METHODS AND RESULTS: Of 86 samples analysed, 28 recorded positive for V. parahaemolyticus by conventional microbiological method, while 53 were positive by the toxR-targeted PCR, performed directly on enrichment broth lysates. While one sample of molluscan shellfish was positive for tdh gene, trh gene was detected in three enrichment broths of molluscan shellfish. CONCLUSIONS: Direct application of PCR to enrichment broths will be useful for the rapid and sensitive detection of potentially pathogenic strains of V. parahemolyticus in seafoods. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus is an important human pathogen responsible for food-borne gastroenteritis world-wide. As, both pathogenic and non-pathogenic strains of V. parahaemolyticus exist in the seafood, application of PCR specific for the virulence genes (tdh & trh) will help in detection of pathogenic strains of V. parahaemolyticus and consequently reduce the risk of food-borne illness.     

多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌

设计引物和探针,优化多重实时PCR条件,以同时检测霍乱弧菌霍乱毒素基因ctxA、副溶血弧菌种特异性基因gyrB和耐热肠毒素基因tdh。该多重实时PCR方法检测产毒素性的O1群(3株)和O139群(44株)霍乱弧菌菌株、不产毒素的O1群(12株)和O139群(6株)及非O1非O139群(7株)霍乱弧菌菌株的ctxA,阳性和阴性结果与普通PCR检测结果100%符合;检测副溶血弧菌种特异性gyrB,116株副溶血弧菌均阳性,而9株其它细菌和72株霍乱弧菌均阴性;检测tdh的阳性和阴性结果也与普通PCR结果完全一致。另外还建立了检测副溶血弧菌菌株trh1和trh2的单重实时PCR方法。     

Detection of Vibrio parahaemolyticus in cockle (Anadara granosa) by PCR

This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.     

Detection of the tdh and trh genes in Vibrio parahaemolyticus isolates in fish and mussels from Middle Black Sea Coast of Turkey

Aim:  The aim of this study was to demonstrate the occurrence of potential pathogenic Vibrio parahaemolyticus in seafoods using DNA-based techniques in comparison with bacteriological methods.
Methods and Results:  From 120 fresh and processed fish and mussel samples collected from Middle Black Sea, 32 isolates were identified as V. parahaemolyticus by bacteriological methods and confirmed by tl gene-based conventional PCR. Of them, 13 isolates were found positive for only tdh gene, six isolates for only trh gene and 13 isolates for both genes by multiplex PCR.
Conclusions:  It is the first report demonstrating the presence of potential pathogenic V. parahaemolyticus isolates from the Black Sea seafoods by PCR detection of tl , trh and tdh genes that was found more rapid than bacteriological methods.
Significance and Impact of the Study:  This study confirmed the previous reports that characterization of potential pathogenic V. parahaemolyticus isolates based on the PCR techniques was reliable and cost-effective. These results suggest that molecular detection methods should be included in Turkish Standards of seafood control in addition to bacteriological methods.     

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

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 >10(4) 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.     

Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10(4) organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.     

结合流式细胞仪检测技术的菌体原位PCR扩增

建立一套原位PCR检测方法 ,联合流式细胞仪作为检测工具 ,作为基因水平转移研究中的基因监控手段。通过常规PCR反应以确定靶基因的基本扩增参数 ;细菌菌体经过多聚甲醛PBS液固定和溶菌酶处理后进行原位PCR扩增 ,产物洗涤后迅速用流式细胞仪进行荧光检测 ,并辅以荧光显微镜镜检。扩增样品在荧光显微镜的蓝光激发下发出明亮的黄绿色荧光 ,与空白对照中的无扩增菌体的自发荧光可明显区分。流式细胞仪检测结果也显示 ,阴性菌与阳性菌的荧光强度有明显区别。完成了对目标细菌的原位PCR扩增 ,并成功地应用流式细胞仪对原位PCR扩增菌体实施了检测。由此表明isPCR 流式细胞仪检测技术在细菌致病基因原位监控上的应用前景     

副溶血弧菌海产品分离株tdh基因及其临近区域结构分析

摘要：【目的】初步探索副溶血弧菌海产品分离株tdh基因区域的结构特征。【方法】采用长距离PCR和基因步移技术进行tdh基因侧翼序列扩增,测序验证后拼接成疑似毒力基因片段,将所获序列与NCBI数据库进行比较,初步明确tdh基因侧翼序列的结构与功能。【结果】海产品分离株ZS34与参考菌株 RIMD2210633的tdh基因区域（VPA1310-VPA1327）结构基本一致,核苷酸同源性达98.3%;而FJ14与WZ64株基因组中的tdh基因均与tdh3的同源性最高,在基因组中的位置也不同于ZS34株和参考菌株     

基于分子马达生物传感器技术的副溶血性弧菌分子分型方法的初步研究

[目的]建立基于分子马达技术的简便快速的分子分型方法,对携带和非携带毒力基因的副溶血性弧菌进行快速分类.[方法]以F0F1-ATPase为核心构建分子马达,以副溶血性弧菌毒力基因tdh、trh和种特异性基因tlh、toxR为靶基因设计4个探针.通过生物素-亲和素系统将探针与分子马达连接构建F0F1-ATPase分子马达生物传感器,对10株副溶血性弧菌分离株进行分类,并与PCR-电泳-凝胶成像结果进行比较;同时对生物传感器的检测灵敏度和特异性进行研究.[结果]10株试验菌株中10株tdh阳性,0株trh阳性,而10株菌都携带tlh和toxR,与PCR-电泳-凝胶成像结果一致;分子马达生物传感器的最低检测限为1 pg/反应体系,且能够对副溶血性弧菌特异性识别,PCR-电泳-凝胶成像方法的最低检测限为10 pg/PCR反应体系.[结论]建立了基于分子马达的分子分型方法,能够对副溶血性弧菌的致病性进行快速诊断,检测灵敏度比PCR-电泳-凝胶成像方法高了10倍,而且特异性非常高.该方法简便、快速、省时、省力,适用于地方疾控部门和口岸检疫部门的基层实验室开展副溶血性弧菌监测和流行病学溯源工作.     

Genome sequence of the clinical O4:K12 serotype Vibrio parahaemolyticus strain 10329

Vibrio parahaemolyticus is the leading cause of food-borne illnesses worldwide. Here, we report a draft genome of V. parahaemolyticus strain 10329 of the O4:K12 serotype. It belongs to the main U.S. West Coast clonal complex of V. parahaemolyticus (sequence type 36 [ST36]) causing oyster-associated human illness. It contains the virulence determinants tdh and trh but appears to infect at much lower doses than V. parahaemolyticus strains with these same determinants from other areas, such as the U.S. Gulf and Atlantic coasts.     

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan

Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh 2 gene, one of the two tdh genes ( tdh 1 and tdh 2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh 1– and tdh 2– lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh 2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh 2 and tdh 1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh 2 promoter; they differ at position −34 but have the same base at position −24 as the tdh 2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.     

Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.