沙门氏菌、金黄色葡萄球菌、志贺氏菌、单增李斯特菌选择性共增菌培养基的研制

目的：研制一种对沙门氏菌（Salmonella）、金黄色葡萄球菌（Staphylococcus aureus）、志贺氏菌（Shigella）和单增李斯特菌（Listeria monocytogens）的选择性共增菌培养基（SSSL培养基）。方法：挑选添加成分进行单因素试验,确定SSSL培养基的成分及配比,采用平板计数法验证SSSL培养基的增菌效果。结果：确立了SSSL培养基配方,目标菌在SSSL增菌培养基中培养8 h后,菌体浓度都达到了105~106CFU/mL,而且抑制非目标菌的生长。结论：SSSL培养基能用于沙门氏菌、金黄色葡萄球菌、志贺氏菌和单增李斯特菌选择性共增菌,可望与多种检测方法联用,以提高检测率和准确性。     

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。     

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。     

一种能同时富集沙门氏菌、金黄色葡萄球菌和单增李斯特菌的培养基

摘要：【目的】设计制备一种能够同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的复合增菌肉汤。【方法】挑选合适的添加剂进行单因素实验,确定增菌肉汤的成分及配比,采用平板计数法及三重荧光PCR技术验证肉汤的增菌效果。【结果】结果得到一种能同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的选择性增菌肉汤（SSL）,经验证SSL可使得3种目标菌以相对一致的速度进行富集,经过37℃ 150r/min 振荡培养24h后,菌体浓度到达107~108CFU/mL,非目标菌生长受到抑制。应用荧光PCR扩增样品,可同时得到3种     

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。     

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。     

海洋蛭弧菌的分离鉴定及其对副溶血弧菌的作用

蛭弧菌广泛存在于自然水体, 具有噬菌的特性, 对水体中细菌数量控制具调节作用。以副溶血弧菌为宿主菌, 利用双层琼脂法, 从海洋水体中分离出15株具有噬菌作用的细菌, 对形成噬菌斑能力最强的1株菌株进行特异性16S rDNA扩增, 确认为蛭弧菌, 命名为Bd-M1。Bd-M1对大多数海水养殖动物病原菌有裂解作用, 裂解率在90%(20/22)以上, 模拟水环境实验发现, 蛭弧菌对副溶血弧菌有较强的裂解和净化作用, 102 h内能使副溶血弧菌从3.0′108 CFU/mL下降到8.7×103 CFU/mL。动物实验表明蛭弧菌能有效预防对虾弧菌病的发生, 表明蛭弧菌有望成为水产动物疾病防治的一种有效的生物制剂。     

一种能同时富集沙门氏菌、金黄色葡萄球菌和单增李斯特菌的培乔基

[目的]设计制备一种能够同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的复合增菌肉汤.[方法]挑选合适的添加剂进行单因素实验,确定增菌肉汤的成分及配比,采用平板计数法及三重荧光PCR技术验证肉汤的增菌效果.[结果]结果得到一种能同时富集沙门氏菌、金黄色葡萄球菌及单增李斯特菌的选择性增菌肉汤(SSL),经验证SSL可使得3种目标菌以相对一致的速度进行富集,经过37℃ 150 r/min振荡培养24 h后,菌体浓度到达10~7～10~8 CFU/mL,非目标菌生长受到抑制.应用荧光PCR扩增样品,可同时得到3种目标菌的扩增曲线.在710份实际样品检测中,无假阳性及假阴性报告.[结论]研究结果表明,SSL肉汤可用于沙门氏菌、金黄色葡萄球菌及单增李斯特菌的共增菌,可用于多重PCR检测的前增菌.     

A multipathogen selective enrichment broth for simultaneous growth of Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae

A selective enrichment broth (SVV) was formulated to allow concurrent growth of Salmonella spp., V. parahaemolyticus, and V. cholerae. Potassium tellurite and sodium citrate were added as the inhibitors, while glucose, mannitol, anhydrous sodium sulfite and sodium pyruvate were employed as the growth-promoters. When mixed in equal or varied proportions, the target pathogens in SVV had a great accumulation (10(5)-10(8) CFU/ml) and effectively inhibited the growth of competitive microflora. In the artificially contaminated samples, a high recovery of these 3 target pathogens was obtained in SVV. Finally, Salmonella spp., V. parahaemolyticus, and V. cholerae were detected from 608 suspicious food samples by SVV with real-time PCR, and no false-positive or -negative results were reported. In summary, SVV has been shown to be a suitable broth for the simultaneous detection of the 3 pathogens by multipathogen detection on a single-assay platform.     

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.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.     

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.     

Helical growth and the curved shape of Vibrio cholerae

The curved, comma, or bent shape of Vibrio cholerae is attributed to, and explained by, the normal helical growth of the cell. The comma-like shape of V. cholerae is not due to an asymmetrical positioning of peptidoglycan such that some chains of peptidoglycan are placed so they are more spread out on one side of the cell and squeezed together on the other side.