四重PCR检测副溶血性弧菌及其毒力基因方法的建立

【目的】建立同时检测副溶血性弧菌tox R、tdh、trh、tlh基因的四重PCR快速检测方法。【方法】分别以副溶血性弧菌的tox R、tdh、trh、tlh 4个基因为靶基因,设计4对特异性引物,对4对引物浓度和退火温度进行优化,获得最佳引物比例和扩增条件,建立快速检测致病性副溶血性弧菌的四重PCR体系。通过特异性验证、灵敏度验证以及模拟样品检测进行方法确认。【结果】四重PCR体系扩增条带与预期相符,即115 bp(tox R)、244 bp(tdh)、418 bp(trh)、759 bp(tlh)4个目的条带;用74株副溶血性弧菌和37株非目标菌的测试结果表明,所建立的方法有良好的特异性。该方法对模板DNA的检测灵敏度为50μg/L,纯培养物的检测灵敏度为6.7×103 CFU/m L;副溶血性弧菌含量为1.36 CFU/g的人工模拟样品增菌6 h后,tox R、tlh、tdh、trh 4个基因可同时被检出。【结论】该方法可实现同时检测携带tox R、tdh、trh、tlh 4种基因的副溶血性弧菌,对开展致病性副溶血性弧菌的检测研究具有一定现实意义。     

副溶血性弧菌食物中毒及腹泻菌株III型分泌系统的检测及分析

目的 了解副溶血性弧菌食物中毒和临床腹泻株III型分泌系统的分布以及耐药特征。方法 对食物中毒和临床腹泻分离到的共21株副溶血性弧菌进行毒力基因tdh、trh、T3SS1、T3SS2α、T3SS2β和toxR检测,并用VITEK 2 compact全自动微生物鉴定系统进行了耐药性分析。结果 21株菌株中tdh＋/trh－占90.48%（19/21）, tdh－/trh＋和tdh－/trh－分别占4.76%、4.76%,未检测到tdh＋/trh＋菌株。T3SS1广泛存在于所有菌株中。T3SS2α存在于tdh＋/trh－菌株,T3SS2β存在于tdh－/trh＋菌株。1株食物中毒菌株毒力基因携带情况为tdh－/trh－/T3SS2α－/T3SS2β－。21株副溶血性弧菌对阿莫西林、头孢吡肟、抗菌素B、庆大霉素、环丙沙星和复方新诺明敏感,对氨苄西林完全耐药。结论 食物中毒和临床腹泻分离到的菌株大多携带tdh基因,T3SS2α与tdh相关,而T3SS2β则存在于trh＋菌株。未携带tdh和trh基因的食物中毒分离株表明副溶血性弧菌不仅仅依赖TDH和TRH发挥毒力作用,其致病机制具有多样性和复杂性。     

2012年上海地区副溶血性弧菌血清分型和毒力基因携带状况研究

为了解2012年上海地区副溶血性弧菌人源株和食源株的优势血清型及其毒力基因携带状况,本研究收集了2012年从上海市15个区(县)腹泻患者和食品监测中分离的副溶血性弧菌株,进行血清分型,并采用聚合酶链反应(PCR)检测tdh和trh基因。结果显示,854株副溶血性弧菌中,88.1%为血清可分型,89.8%为产毒株。O3∶K6、O4∶K8、O1∶K25、O4∶K68、O4∶K9、O1∶K36、O3∶K29为上海地区可分型人源株的优势血清型(93.8%),其中O3∶K6最多,达56.2%。副溶血性弧菌全部分离株的月份分布显示出聚集趋势,7~8月为高峰期。O4∶K9和O1∶K36血清型菌株的月份分布与其他优势血清型菌株不同,未表现出明显聚集趋势。食源株无明显优势血清型,且与人源株分布不同。人源株产毒株构成(95.6%)高于食源株(5.5%)。人源株优势血清型产毒株构成(99.9%)高于非优势血清型(71.1%)。血清可分型人源株的tdh携带率(97.5%)高于不可分型人源株(67.6%),血清可分型人源株的trh携带率(0.8%)低于不可分型人源株(42.6%)。结果提示,副溶血性弧菌血清型分布与历史数据相比变化较大,血清型与毒力基因携带呈一定程度关联,且人源株与食源株在血清型和毒力基因携带上具有分离现象。因此,在副溶血性弧菌的监测与检测中应充分考虑血清分型和毒力基因的重要性。     

实时荧光PCR检测水产品中副溶血性弧菌

目的探索副溶血性弧菌快速检测法,应用于日常监测及食物中毒的快速查源。方法用副溶血性弧菌实时荧光试剂盒对水产品样本进行检验,以副溶血性弧菌toxR基因为靶序列,设计1对引物和探针,采用热裂解法提取DNA。结果实时荧光PCR从42份水产品样品的增菌液中检出13份样品副溶血性弧菌阳性,与传统培养法相比一致性极好（K=0.943,K〉0.75）。结论实时荧光PCR方法在副溶血性弧菌的检验方面较传统方法具有快速、灵敏、特异性强等优势,具有广阔的应用前景。     

EMA-LAMP方法快速检测鉴别副溶血性弧菌

建立将DNA染料EMA(ethidium bromide monoazide)结合环介导等温扩增技术(loop-mediated isother-mal amplification,LAMP)的方法(EMA-LAMP),用于检测鉴别副溶血性弧菌(Vibrio parahaemolyticus)死/活菌细胞。针对副溶血性弧菌不耐热溶血素基因tlh(thermolabile hemolysin)特异性序列的6个位点设计4条引物及2条环引物,进行检测。结果表明,浓度为8.0μg/mL或更高浓度的EMA,至少经25 min的曝光处理,能够有效抑制浓度为1×108cfu/mL的副溶血性弧菌死细胞的扩增,而对用相同浓度EMA处理的副溶血性弧菌活细胞扩增没有影响。经EMA处理,含有不同比例的副溶血弧菌死细胞和活细胞的混合液中,活菌的最小检测限为1.0×102cfu/mL。EMA-LAMP方法比EMA-PCR方法区分死活细胞中的活细胞更为有效,是一种能够快速、灵敏且更为有效鉴别副溶血性弧菌死活细胞的新方法。     

基于颜色判定的环介导恒温扩增法快速检测副溶血性弧菌

利用DNA环介导恒温核酸扩增法（LAMP）针对副溶血性弧菌特异基因tlh基因设计4条引物,通过引物特异性识别tlh基因上的6个独立区域来快速检测副溶血性弧菌.LAMP反应的过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果.实时浊度仪监测反应结果表明,LAMP反应在60～65℃恒温条件下50min内完成;如果在反应前添加羟基萘酚兰（HNB）,蓝色的阳性结果很明显区别于紫色阴性结果;LAMP方法的最低检出限为9.74pg/μL,PCR方法最低检出限为97.4pg/μL,LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍,且具有良好的特异性.LAMP方法用于快速检测副溶血性弧菌具有检测过程简单、实验装置简便、反应结果肉眼可辨别、灵敏度高和特异性强的特点,所以LAMP方法检测副溶血性弧菌特别适合用于现场和基层检疫及医疗单位的快速诊断.     

副溶血性弧菌分子标志基因研究概况

副溶血性弧菌是重要的食源性致病菌,其中,O3:K6血清型是1996年后导致多个国家多起食物中毒暴发的病原菌。中国1992-2001年的统计数据表明,由副溶血性弧菌引起的胃肠炎占由微生物引起的食源性疾病暴发的31.1%。副溶血性弧菌环境株大部分是非致病性的,而临床株则能产生耐热直接溶血素、耐热相关溶血素以及其它毒力因子。本文综述了3种重要的副溶血性弧菌的分子标志物,包括种特异性基因、毒力基因以及大流行菌群特异基因,旨在为研究者们针对性的选取基因开展快速检测副溶血性弧菌和鉴别其致病因子的研究提供参考依据。     

toxR基因作为荧光定量PCR靶基因设计TaqMan探针快速检测副溶血弧菌

以toxR基因为靶基因,通过优化反应条件建立了快速检测副溶血弧茵的TaqMan实时荧光PCR方法.特异性试验表明,该方法能选择性检测副溶血弧茵,而与金黄色葡萄球菌、沙门氏菌、单增李斯特杆菌等多种常见的食源性病原茵没有交叉反应:灵敏度试验表明,该方法最少可检测到25个拷贝的toxR基因重组质粒,对纯培养物和模拟食品样品直接检测的灵敏度分别为21 cfu/mL和210 cfu/g;重复性试验表明,同一样品于试验内及试验间的变异系数分别为0.9%和1.3%:所制作的标准曲线在2.5 × 101～2.5 × 106拷贝数之间有较好的线性关系,能对副溶血孤菌进行准确的定量分析.结果表明,本研究所建立的副溶血弧菌实时荧光PCR检测方法具有特异性好,灵敏度高、重复性好的特点,能进行定量检测,而且检测时间从核酸抽提到出实验结果仅需要3 h.是快速检测副溶血弧菌的有效手段.     

四种食源性致病弧菌多重实时荧光定量PCR快速检测体系的建立

【背景】弧菌在全球范围内严重威胁人类健康,副溶血性弧菌(Vibrio parahaemolyticus)、霍乱弧菌(Vibrio cholerae)、拟态弧菌(Vibrio mimicus)和创伤弧菌(Vibrio vulnificus)与食用生的或未煮熟的海鲜所引起的胃肠道感染和败血症有关,因而备受关注。【目的】建立一种实时荧光定量PCR方法,用于检测副溶血性弧菌、霍乱弧菌、拟态弧菌和创伤弧菌,以提高检测效率和准确性。【方法】基于副溶血性弧菌和拟态弧菌的toxR基因、霍乱弧菌的ompW基因、创伤弧菌的vvhA基因设计特异性引物和探针,通过优化反应体系和条件建立四重实时荧光定量PCR体系。【结果】该实时荧光定量PCR方法检测限均为10 copies/µL,扩增效率均在100%左右;在特异性试验中,分别运用多重实时荧光定量PCR和常规PCR对目的菌基因组、非目的菌基因组和空白对照进行扩增,结果均只有目的弧菌扩增明显,表明本方法有良好的特异性;在抗干扰实验中,高浓度弧菌不干扰低浓度弧菌检测;每个浓度梯度进行3次重复实验,每组变异系数均小于1.5%,表明本方法重复性好。【结论】建立的多重实时荧光定量PCR方法可快速、特异地实现对副溶血性弧菌、霍乱弧菌、拟态弧菌和创伤弧菌的检测。     

副溶血性弧菌CRISPR的检测及其结构分析

【目的】检测副溶血性弧菌(Vibrio parahaemolyticus,简称VP)中规律成簇间隔的短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR),并对不同来源的VP中CRISPR位点的结构多样性进行分析。【方法】根据CRISPR DB数据库中公布的VP中确定的CRISPR结构序列CRISPR-1及文献中新发现的疑似CRISPR结构序列CRISPR-2设计引物,对不同来源的79株VP进行PCR扩增。利用CRISPR Finder分析CRISPR结构,采用生物信息学方法对不同来源VP的CRISPR位点结构多样性进行比较分析。【结果】79株VP中CRISPR-1的检出率为92.41%,CRISPR-2的检出率为96.20%,同时具有这2个位点的菌株占总数的89.87%,只有1株菌被检出不含有任何位点。分别比较不同来源的菌株CRISPR-1、CRISPR-2位点的重复序列发现不存在序列差异,而临床菌株的这2个CRISPR位点在间隔序列上比环境分离菌株存在更多的变异。2个CRISPR位点根据间隔序列的不同在VP中一共组成8种CRISPR谱型(编号A-H),除F谱型外,A-E、G谱型均只在临床分离菌株中发现,而在环境分离菌中还发现不含任何位点的H型。【结论】CRISPR在VP中普遍存在。环境分离菌株与临床分离菌株中CRISPR的结构存在差异。     

Vibrio virulence genes in fishes collected from estuarine waters in Italy

Aims: The aim of this study was to investigate the presence of Vibrio vulnificus and potentially pathogenic strains of Vibrio parahaemolyticus in mullets collected from estuarine environment in Italy. Methods and Results: Two hundred and ninety‐five mullets were analysed by culture using the selective medium thiosulfate citrate bile salt sucrose agar, during a monitoring period of 2 years (2008–2009). Presumptive Vibrio colonies were initially identified by using biochemical tests, and strains identified as V. parahaemolyticus and V. vulnificus were subsequently examined by PCR for the presence of species‐specific and virulence genes (toxR, trh, tdh and vvh). V. parahaemolyticus was found in 55% (162/295) of fishes and V. vulnificus in 1% (3/295) with a higher presence in summer months. The trh+/tdh? strains were detected in 16% (47/295) of samples and only one strain resulted trh+/tdh+. One of the V. parahaemolyticus trh+ strains isolated belonged to the O1:KUT (K untypeable), a serotype recently associated to gastroenteritis in Italy. Conclusions: This is the first report demonstrating a high percentage of potential pathogenic V. parahaemolyticus trh+ strains in estuarine fishes of the Mediterranean area. Significance and Impact of the Study: These findings indicate the potential human health risk associated with the presence of pathogenic Vibrio spp. in wild fishes.     

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.     

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.     

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.     

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.     

2013‒2015年杭州地区急性腹泻患者来源副溶血弧菌毒力基因及耐药性调查

目的 对2013‒2015年杭州地区腹泻患者副溶血弧菌（Vibrio parahaemolyticus）毒力基因的携带和耐药进行调查研究。方法 可疑菌株用全自动细菌鉴定仪（VITEK2 Compact,生物梅里埃,法国）进行鉴定,用PCR方法检测其8种毒力基因,用纸片扩散法检测13种抗生素的敏感性。结果 120株副溶血弧菌中,tdh+或trh+的致病菌株占95.0%（114株）,tdh-且trh-的非致病株占5.0%（6株）。所有120株副溶血弧菌均含有T3SS1基因,T3SS2α基因主要存在于tdh+/trh-的菌株中,T3SS2β存在于trh+的菌株中,2株tdh-的菌株首次检出T3SS2α基因。本研究中大流行菌株占64.2%（77株）,非大流行菌株占35.8%（43株）。多达65.8%的副溶血弧菌对氨苄西林耐药,而90.0%以上的副溶血弧菌对其他抗生素包括喹诺酮类、第三代头孢菌素类、氨基糖苷类、四环素类和磺胺类抗菌药物较敏感。结论 杭州地区腹泻来源的副溶血弧菌多为大流行菌株,对常见抗菌药物敏感性高,但耐药率有所上升,要加强监测并引起临床重视。     

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     

Genetic diversity among Norwegian Vibrio parahaemolyticus

Aims: To examine the variability among environmental Vibrio parahaemolyticus (including trh+ isolates) from Norway, and to compare these to clinical isolates and isolates from imported foods. Methods and Results: A total of 246 V. parahaemolyticus were successfully digested with NotI, and the fragments were separated by pulsed field gel electrophoresis (PFGE). The isolates could be divided into 72 clusters and 103 pulsotypes. Eleven clusters contained 4–31 environmental isolates, and the isolates within these clusters greatly varied with respect to origin. None of the trh+ and /or tdh+ isolates clustered with trh?/tdh? isolates. The trh+ environmental isolates included in the study belonged to two separate clusters. A subset of isolates was serotyped, and great serotype diversity was observed among the environmental V. parahaemolyticus. The clinical isolates included O3:K6 and O3:KUT, and these were identical or related to a pandemic reference strain by PFGE. Conclusions: Environmental V. parahaemolyticus (including trh+) were genetically diverse, but certain variants occurred throughout the coastal environment, and some were persistent over time. Significance and Impact of the Study: Although trh+ V. parahaemolyticus persisted in the Norwegian environment, no evidence indicated that indigenous isolates have caused disease.