Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus

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.     

荧光染料EMA在实时荧光PCR定量检测海产品中致病性副溶血弧菌活细胞中的应用

将荧光染料--叠氮溴乙锭(ethidium bromide monoazide,EMA)与实时荧光-聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)技术相结合,对其在RT-PCR定量检测海产品中致病性副溶血弧菌活细胞中的应用进行研究。纯培养条件下,在2.2×102～2.2×107CFU范围内细胞数的常用对数值与Ct值之间呈严格的负相关性,并且不添加EMA时的检测灵敏度(22CFU)略大于添加EMA(2.2×102CFU)时的检测灵敏度;人工污染牡蛎样品,利用RT-PCR和EMA RT-PCR以及平板计数法分别进行副溶血弧菌定量检测,结果表明EMA RT-PCR更接近于平板计数的结果,单纯RT-PCR定量的结果偏大;在采集的45份海产品样品中,不经富集培养,利用EMA RT-PCR方法仅有一份牡蛎样品呈阳性,牡蛎样品污染程度为114CFU/g。该方法能够快速、灵敏、准确地进行海产品中致病性副溶血弧菌活细胞的定量检测,具有很好的应用价值。     

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.     

Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA

PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.     

Levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene-positive organisms in retail seafood determined by the most probable number-polymerase chain reaction (MPN-PCR) method

The incidence and levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene (tdh)-positive organisms in retail seafood were determined. The most probable number-polymerase chain reaction (MPN-PCR) method using a PCR procedure targeting the species-specific thermolabile hemolysin gene (tlh) and tdh was used to determine the levels of V. parahaemolyticus and tdh-positive organisms, respectively. In seafood for raw consumption, V. parahaemolyticus was found in four (13.3%) of 30 fish samples, 11 (55.0%) of 20 crustacean samples, and 29 (96.7%) of 30 mollusc samples. Levels of V. parahaemolyticus were below 10(4) MPN/100 g in all fish and crustacean samples tested. However, they were above 10(4) MPN/100 g in 11 (36.7%) of the 30 mollusc samples. In all seafood for raw consumption, the level of tdh-positive organisms was below the limit of detection (< 30 MPN/100 g). In seafood for cooking, V. parahaemolyticus was found in 15 (75.0%) of 20 fish samples, nine (45.0%) of 20 crustacean sample, and 20 (100%) of 20 mollusc samples. Levels of V. parahaemolyticus were above 10(4) MPN/100 g in only three (15.0%) and one (5.0%) of the 20 fish and 20 crustacean samples, respectively. However, they were above 10(4) MPN/100 g in 18 (90.0%) of the 20 mollusc samples. In seven (35.0%) of the 20 mollusc samples, tdh-positive organisms were found and their levels ranged from 3.6x10 to 1.1 x 103 MPN/100 g. From four of seven tdhpositive samples, tdh-positive V. parahaemolyticus was isolated.     

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1 cfu/mL and 10 cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.     

多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌

目的建立检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌的多重荧光定量PCR体系。方法针对副溶血性弧菌tlh基因,沙门菌Ompc基因和单增李斯特菌hly基因设计引物和Taq Man探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血性弧菌、沙门菌和单增李斯特菌。结果副溶血性弧菌、沙门菌和单增李斯特菌可得到特异性扩增,而共存于海产品中的其他细菌均未见扩增曲线。敏感性试验显示,该体系对副溶血性弧菌、沙门菌和单增李斯特菌的最低检测限分别为72、40、80 cfu/ml。对舟山采集的150份样品进行检测,检出32份副溶血性弧菌、11份沙门菌、5份单增李斯特菌,与国标法检测结果一致。结论本研究建立的基于Taq Man探针的多重荧光定量PCR检测方法可以特异、灵敏、简单快速地实现对海产品中副溶血性弧菌、沙门菌和单增李斯特菌的检测。     

Evaluation of an alkaline phosphatase-labeled oligonucleotide probe for the detection and enumeration of the thermostable-related hemolysin (trh) gene of Vibrio parahaemolyticus

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase-labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.     

副溶血性弧菌PMA-LAMP方法的建立

副溶血性弧菌(Vibrio parahemolyticus)是一种常见的食源性致病菌,在海鲜等食物中检出率很高,食用没有煮熟的带菌食物或者腌制品,极易引起食物中毒。目前用以检测副溶血性弧菌的快速检测方法都不能区别死活菌,容易出现假阳性结果。本研究基于DNA结合染料叠氮溴化丙锭(propidium monoazide,PMA),利用荧光染料钙黄绿素(Calcein)的特性,建立了一种实时的可视化环介导等温扩增方法(Loop-mediated isothermal amplification,LAMP),并与q PCR方法进行比较。实时可视化LAMP方法的反应结果,既可以肉眼直接观测,也可以借助荧光分析设备实时监测,并对半定量分析进行初探,为之后向定量分析深入研究打下基础。实验结果表明,实时可视化PMA-LAMP方法的灵敏度可达5.0×102 CFU/m L,模拟食样灵敏度为1.9×102 CFU/m L,与PMA-q PCR方法结果一致。除去前增菌时间,整个检测过程只需2 h,且准确率高,为副溶血性弧菌快速检测的开展提供了有效的技术支持。     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

北部湾茅尾海副溶血性弧菌的分离鉴定与致病性分析

为解析北部湾海域及水产品中副溶血性弧菌的多样性特征与安全风险,本研究采集了北部湾茅尾海养殖区域海水和水产经济动物样品,利用硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基(TCBS)对所采样品进行海洋弧菌的分离和纯化,共分离获得109株疑似弧菌菌株。通过16S rDNA和特异功能基因toxR的PCR扩增并测序鉴定,共检出副溶血性弧菌20株,检出率为18.3%。此外,通过系统发育分析还发现副溶血性弧菌的toxR和tdh基因序列都存在水平基因转移现象,呈现出较大的多样性。对20个副溶血性弧菌菌株的毒力基因tdh进行分析,结果表明有4株携带了tdh毒力基因,检出率为20%,易引起食物中毒,对公共卫生造成的威胁较大。因此,本研究建议采用PCR技术开展副溶血性弧菌特异种属基因和毒力基因检测,准确评估北部湾区域海水及其水产品的卫生安全性,降低爆发水产养殖业病害和食源性疾病的风险。     

巢式PCR快速检测海产品中的副溶血弧菌

副溶血弧菌是一种世界范围性的食源性致病菌,食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法,通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析,筛选出了一个副溶血弧菌特异性的标记基因-VP1331,根据该基因建立了副溶血弧菌的巢式PCR快速检测方法,并评估了其特异性、敏感性和稳定性。实验结果表明,该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段,而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明,初始菌液浓度为25.7 CFU/100 mL时只需经过2 h的增菌培养即可检出。上述结果表明,VP1331基因可以作为副溶血弧菌种特异性标记,本方法可以用于污染海产品中该菌的检测与鉴定。     

Comparison of a Chromogenic Medium with Thiosulfate-Citrate-Bile Salts-Sucrose Agar for Detecting Vibrio parahaemolyticus

ABSTRACT: The widely used most probable number (MPN) method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus on the thiosulfate-citrate-bile salts-sucrose agar (TCBS). Presumptive positive colonies grown onTCBS need to be confirmed with lengthy biochemical tests. This study compared a chromogenic medium, Bio-Chrome Vibrio medium (BCVM), with TCBS for detecting V. parahaemolyticus in seawater, sediment, and oysters using a 3-tube MPN method. Among the 296 samples tested, 136 and 92 samples produced presumptive positive results on TCBS and BCVM, respectively. Biochemical tests and a multiplex polymerase chain reaction (PCR) assay confirmed 74 of 83 samples that were presumptive positive on both TCBS and BVCM as V. parahaemolyticus . Although false-positive results were reported when either medium was used, there were 62 reported for TCBS whereas only 15 were reported for BCVM. The specificities of TCBS and BCVM for V. parahaemolyticus detection were determined to be 77% and 94%, respectively. The accuracies of detecting V. parahaemolyticus were 54% for TCBS and 84% for BCVM. The Bio-Chrome Vibrio medium can be used in the MPN method to reduce the number of biochemical tests needed for V. parahaemolyticus confirmation.     

副溶血弧菌的SYBR Green Ⅰ实时定量PCR检测方法建立

基于副溶血弧菌gyrB基因保守序列设计1对特异性引物,建立SYBR GreenⅠ实时定量聚合酶链式反应(polymerase chain reaction,PCR)检测副溶血弧菌的方法。SYBR GreenⅠ实时定量PCR的Tm为90℃,扩增产物的熔解曲线只出现1个单特异峰,无引物二聚体,表明该引物具有较好的特异性;所制作的实时定量PCR扩增标准曲线在2.06×108～2.06×103拷贝数之间有较好的线性关系,相关系数为0.992,能对副溶血弧进行准确的定量分析。该方法检测时间从核酸抽提到结果分析仅需4～5h,且较传统方法敏感、操作简单,可用于针对副溶血弧菌的进出口检验检疫、食品安全检测及该菌引起的水产动物疾病的诊断与分子流行病学调查。     

双层平板直接定性定量法检测海产品中副溶血性弧菌

目的:建立一种直接定量定性检测海产品中副溶血性弧菌的方法。方法:参照国标菌落计数方法,筛选具备计数和显示副溶血性弧菌典型菌落颜色功能的培养基,将人工染菌样品分别置于50、4和-18℃温度下并分别进行测试。结果:在NaCl NA双层平板上副溶血性弧菌菌落呈淡紫色,在人工染菌样品实验中与NaCl NA平板计数无显著差异;NaCl NA双层平板直接定量检测4℃冷藏处理和-18℃冷冻的样品中副溶血性弧菌浓度分别是对照组的68.9%和21.7%。结论:NaCl NA双层平板法具备操作简便、检验周期短、结果稳定、灵敏度高等特点,适合应用于-18~50℃温度环境下放置的海产品中副溶血性弧菌直接定性定量检测,能如实反映样品受污染的情况。     

恒温实时荧光法检测副溶血性弧菌

为了更加准确、快速地检测进出口食品中的副溶血性弧菌,针对副溶血性弧菌的tlh基因设计环介导等温扩增(loop-mediatedisothermal amplification,LAMP)引物,结合ESEQuant tube scanner来对扩增结果进行实时检测。结果显示:该引物扩增效率高、特异性强、灵敏度高(1-10 CFU/反应,约为荧光PCR的100倍)。126株副溶血性弧菌,恒温荧光法检测出126株,荧光PCR的方法检测出121株。恒温实时荧光检测仪与荧光PCR仪类似,均可以对检测结果进行实时监测和熔解曲线分析,表明该恒温实时荧光检测仪可广泛应用于基础实验室的科学研究或经济发展相对落后区域的临床初步诊断。     

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.