不同遗传背景下QsvR调控副溶血弧菌基因表达的转录组分析

【背景】OpaR是副溶血弧菌群体感应系统的核心调控因子;QsvR是AraC家族转录调控因子,与OpaR之间具有相互调控作用;此外,QsvR对基因表达的调控作用受OpaR的影响,但是影响程度并未完全阐明。【目的】探究在野生株(wild-type,WT)和opaR基因突变株(ΔopaR)的遗传背景下QsvR的转录调控元,分析Opa R对QsvR基因表达调控的影响。【方法】分别以WT和ΔopaR为参照,采用Illumina HiSeq测序平台进行比较转录组学研究,分析生物膜形成条件下qsv R基因突变株(Δqsv R)和Δqsv RΔopaR的基因表达情况。【结果】在WT遗传背景下,QsvR共调控1735个基因的转录(调控元1),其中被激活的基因有855个,被抑制的基因有880个;在ΔopaR遗传背景下,QsvR共调控1 187个基因的转录(调控元2),其中被激活的基因有533个,被抑制的基因有654个。调控元1和调控元2之间共有517个重叠基因,且QsvR对绝大多数重叠基因的调控关系相反。基因属性分类(gene ontology, GO)数据库富集分析结果显示,调控元1和调控元2中分别有4...     

水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

目的 建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法 选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果 确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60 ℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×102 CFU/mL,创伤弧菌最低限为4.2×104 CFU/mL。双重PCR对分离株检测符合率达100%。结论 建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。     

群体感应系统核心调控子对副溶血弧菌mshH基因的调控研究

【目的】探究副溶血弧菌群体感应(quorum sensing,QS)系统核心调控子AphA和OpaR对mshH基因的转录调控。【方法】提取特定条件下副溶血弧菌野生株(wild-type,WT)和调控子基因突变株(ΔaphA和ΔopaR)的总RNA,采用实时定量PCR (quantitative real-time PCR,qPCR)研究AphA和OpaR对mshH基因的转录调控关系以及mshH基因的时相依赖性表达特性;将mshH启动子区DNA序列克隆入pHRP309质粒β-半乳糖苷酶基因的上游,构建LacZ重组质粒,并将其转入WT、ΔaphA和ΔopaR中,获得LacZ实验菌株,再通过LacZ报告基因融合实验研究AphA和OpaR对mshH基因的调控关系以及mshH基因的时相依赖性表达特性;PCR扩增mshH上游启动子区DNA序列,并纯化His-AphA和His-OpaR蛋白,通过凝胶阻滞实验(electrophoretic mobility shift assay,EMSA)和DNase I足迹实验,研究体外条件下His-AphA和His-OpaR对靶基因启动子区DNA片段是否具有直...     

副溶血弧菌毒力评价小鼠模型的建立与应用

目的建立用于评价副溶血弧菌毒力的小鼠模型,为研究副溶血弧菌的致病机制奠定基础。方法将适宜浓度的菌液经腹腔感染4～5周龄雌性BALB/c小鼠,观察小鼠的症状及死亡数。结果高盐(2%NaCl)条件下培养的强毒株RIMD2210633,经腹腔感染107CFU的菌量,小鼠存活率为20%～30%,而环境无毒株S251的小鼠存活率为100%。结论建立了评价副溶血弧菌毒力的实验小鼠模型,并应用于不同盐分浓度培养的强毒株与环境无毒株的毒力比较实验。     

副溶血弧菌EMA-PCR检测技术的建立

PCR技术被广泛应用于副溶血弧菌的检测中, 但传统的PCR技术无法区分样品中的死细菌与活细菌, 往往使检测结果出现较高的假阳性。因此, 将叠氮溴乙锭(Ethidium monoazide bromide, EMA)与PCR技术结合, 建立一种快速、准确的副溶血弧菌检测方法。以dnaJ基因为检测副溶血弧菌的靶基因, 分别用副溶血弧菌的纯培养细胞及其基因组DNA作模板进行PCR检测, 灵敏度分别为2.5×104 CFU/mL和6×102 fg/μL。在检测样品前处理过程中加入EMA, 当EMA的浓度小于5 mg/L时, EMA对活菌靶基因的扩增没有明显的抑制; 而终浓度为2 mg/L的EMA, 能有效抑制1×108 CFU/mL副溶血弧菌死菌的扩增。活菌和死菌混合体系的PCR结果表明, EMA-PCR能有效降低副溶血弧菌检测过程中的假阳性。     

副溶血弧菌的致病机制

副溶血性弧菌是引发微生物食源性疾病的首要病原菌,其在临床上主要引起3种疾病,即胃肠炎、伤口感染和败血症。经过多年的研究,人们对副溶血性弧菌的致病机制有了一定的认识。我们着重介绍副溶血性弧菌的主要毒力因子、毒力基因表达调控及常用的毒力表型研究方法。     

ToxR负调控副溶血弧菌中c-di-GMP的合成

副溶血弧菌(Vibrio parahaemolyticus)是世界范围内引起海产品相关食物中毒的主要致病菌,具有很强的生物膜形成能力。ToxR是一种膜结合调控蛋白,对副溶血弧菌生物膜形成具有一定的调控作用,但具体机制尚未见报道。c-di-GMP是一种普遍存在于细菌中重要的第二信使,参与调控细菌的多种生物学行为包括生物膜的形成。本文探究ToxR对副溶血弧菌中c-di-GMP代谢的调控作用。利用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定副溶血弧菌野生株(wild-type,WT)和toxR突变株(ΔtoxR)中c-di-GMP水平的差异。挑选c-di-GMP代谢相关基因scrA、scrG和vpa0198为进一步研究的靶标,采用实时定量qPCR实验检测靶基因在WT和ΔtoxR中的转录水平差异;将靶基因调控区DNA序列克隆入pHRP309质粒中无启动子的β半乳糖苷酶基因上游,采用lacZ报告基因融合实验进一步研究ToxR对靶基因的转录调控关系;将重组质粒分别导入含有pBAD33或pBAD33-toxR的EC100lpir中,采用lacZ报告基因融合实验研究ToxR是否能在异体宿主中调控靶基因的表达;PCR扩增靶基因上游调控区DNA序列,并纯化His-ToxR蛋白,用凝胶阻滞实验(electrophoresis mobility shift assay,EMSA)研究His-ToxR与靶基因启动子区DNA序列是否具有结合作用。ELISA结果显示ΔtoxR中c-di-GMP含量显著性高于WT中的,说明ToxR抑制c-di-GMP的产生;实时定量qPCR结果表明WT中scrA、scrG和vpa0198的转录水平显著性高于ΔtoxR中的,表明ToxR抑制它们的转录;lacZ报告基因融合实验结果表明ToxR可抑制副溶血弧菌和EC100lpir中scrA、scrG和vpa0198的启动子区活性;EMSA实验显示His-ToxR能特异性地结合到scrA和scrG的上游调控区DNA序列上,而对vpa0198的上游调控区DNA序列无结合作用。综上所述,ToxR通过直接调控相关酶蛋白基因的转录来抑制副溶血弧菌内c-di-GMP的合成,从而有助于精确调控生物膜形成等细菌行为。     

副溶血弧菌LAMP检测方法的建立

副溶血弧菌(Vibrio parahaemolyticus)是一种能引起食源性疾病的重要病原菌。首次将一种新颖的核酸扩增技术-环介导等温扩增技术（Loop-Mediated Isothermal Amplification, LAMP）应用于副溶血弧菌的快速检测。针对副溶血弧菌不耐热溶血毒素基因（tlh）设计四条特异性引物（两条内引物和两条外引物）进行LAMP扩增,对扩增反应进行优化,最佳反应时间为60 min,反应温度为60 ℃。对12种细菌共28株菌进行LAMP扩增,仅14株副溶血弧菌得到阳性扩增结果,证明引物具有很高的特异性。副溶血弧菌基因组DNA和纯培养物的检测灵敏度分别约为90 fg和24 cfu/mL。对模拟食品样品进行直接检测,检测限为89 cfu/g。结果表明,该方法检测副溶血弧菌特异性强、灵敏度高,并且操作简便、检测成本低,1 h即可完成,有望发展成为快速检测副溶血弧菌的有效手段。     

AphA蛋白对副溶血弧菌vopT的转录调控研究

【目的】研究副溶血弧菌AphA对vopT的转录调控机制。【方法】提取野生株(WT)和aphA突变株(ΔaphA)的总RNA,采用引物延伸实验研究vopT的转录起始位点,并根据产物的丰度差异判断AphA对其调控关系。分别将WT和ΔaphA的总RNA逆转录成cDNA,利用实时定量RT-PCR进一步研究AphA对靶基因的调控关系。将vopT的启动子区克隆入pHRP309质粒的β-半乳糖苷酶基因上游,构建LacZ重组质粒,并将该重组质粒转入WT和ΔaphA中,通过测定并比较两株菌中β-半乳糖苷酶活性的差异来判定AphA对vopT的调控关系。PCR扩增靶基因整个启动子区DNA序列,并纯化His-AphA蛋白,利用凝胶阻滞实验(EMSA)验证His-AphA对靶基因启动子区是否具有直接的结合作用。【结果】vopT只有一个转录起始位点A (?86),且其转录活性受AphA的间接抑制。RT-PCR和EMSA结果显示AphA对vtrA的转录也具有间接的抑制作用。【结论】AphA间接抑制vopT转录,且该间接抑制作用与VtrA无关。     

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.     

Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR

Aims: Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders and is transmitted through ingestion of raw or undercooked contaminated seafood. We used the groEL gene for the species‐specific detection of V. parahaemolyticus from artificially inoculated shellfish, fish and seawater. Methods and Results: The nucleotide sequences of 24 Vibrio and seven non‐Vibrio spp. were compared, and less conserved regions were selected for the designing of primer sets. To detect V. parahaemolyticus specifically, PCR conditions were standardized and tested to evaluate the specificity of primers. A 510‐bp band was appeared only from V. parahaemolyticus by PCR. Notably, the detection was shown to be functional at high annealing temperature above 68°C. The groEL primers detected 100 pg and 1 ng of DNA purified from V. parahaemolyticus culture and artificially infected oyster tissue, respectively. Conclusions: The groEL gene is a potential marker for the species‐specific detection of V. parahaemolyticus and could be used to detect this bacterium in contaminated food by PCR. Significance and Impact of the Study: PCR using primers designed from groEL gene provide an efficient method for the accurate identification of V. parahaemolyticus from contaminated samples.     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

特异性三重PCR快速检测副溶血性弧菌

【目的】建立同时检测副溶血性弧菌gyrase、tdh、trh基因的三重PCR快速检测方法。【方法】将已报道的这3种基因的引物加入一个PCR反应管中,对引物浓度和退火温度进行优化,找到最佳引物比例和扩增条件。通过特异性验证、灵敏度验证以及方法间对比进行方法确认,其PCR产物使用全自动毛细管电泳分析系统进行分析。【结果】仅在91、269、485 bp处分别出现预期DNA扩增条带;纯培养条件下,扩增gyrase、tdh、trh的菌浓度检测限分别为6.6×101、6.6×102和6.6×101 CFU/mL;本底干扰物存在时,扩增gyrase、tdh、trh的菌浓度检测限分别为6.6×103、6.6×104和6.6×103 CFU/mL;模板DNA浓度检测限为1.36μg/L。检测进境海产品时,检测结果和FDA 2004标准结果一致,且更易辨认和判断。【结论】此检测方法的成功建立,为副溶血性弧菌及携带tdh和/或trh基因的致病性副溶血性弧菌的检测提供了一种准确、高效、便捷的分子技术手段。     

用基于TaqMan探针的Real-time PCR技术定量检测副溶血弧菌

副溶血弧菌是一种引起食源性疾病的重要病原菌,传统的鉴定方法费时费力且容易出现假阴性,建立一种定量检测副溶血弧菌基因的方法尤为重要。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RealtimePCR方法。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株非副溶血弧菌均不产生扩增曲线,证明了引物和探针具有很高的特异性。细菌纯培养物品和人工布菌的检测敏感度分别为1CFUPCR反应体系和10CFUPCR反应体系,相关系数均为0.99(r2=0.99),整个试验可在1h内完成。建立的方法可用于海产品中副溶血弧菌的快速定量检测。