霍乱弧菌检测技术研究进展

随着社会的发展,公共卫生及水体质量已得到明显提升,但到目前为止在发展中国家由霍乱疫情导致的死亡率依旧非常高,而霍乱弧菌即为霍乱疫情爆发的病原菌,且霍乱在我国被列为甲类传染病。目前国内外针对霍乱弧菌已经建立了多种有效的检测技术,对控制霍乱暴发作用显著。本文综述了近年来霍乱弧菌检测技术研究新进展,包括微生物学、免疫学、分子生物学等较成熟的检测方法,生物传感器、快速检测技术等新兴检测方法,并总结各种技术优缺点,展望未来霍乱弧菌检测的市场需求。     

LAMP技术用于霍乱弧菌检测的研究

霍乱是由霍乱弧菌感染所引起的,属于甲类传染病.因此,建立一种快速、灵敏、特异的检测方法,对霍乱的预防和控制工作有十分重要的现实意义.通过霍乱弧菌外膜蛋白的ompW基因设计特异引物,建立霍乱弧菌的LAMP检测方法,同时对细菌进行扩增以检测该方法的灵敏度和特异性,并与普通PCR进行比较.实验的全部反应可在2 h内完成,且反应结果可通过肉眼观测直接判定;实验中霍乱弧菌的LAMP检测方法灵敏度是普通PCR的100倍,最低检出下限为10-5;检测霍乱弧菌均为阳性,其它13株非霍乱菌则全部阴性,特异度为100%.结果表明:该方法能快速、灵敏、特异性地检测霍乱弧菌,为快速检测病原微生物起到了很好的借鉴作用.     

梅毒实验室检测方法的研究进展

梅毒是由梅毒螺旋体引起的一种严重危害人类健康的性传播疾病,选择敏感性高、特异性强的检测方法对于梅毒的确证和临床指导应用均具有很重要的意义。当前,有多种梅毒实验室检测方法,其中直接检测法在近十年的临床诊断中仍被经常使用,血清学检测法仍然是目前梅毒实验室诊断的主要方法,以各类PCR技术为代表的梅毒分子生物学检测方法因其具有高度的特异性和敏感性已被广泛应用于临床检验。对梅毒实验室检测技术中直接法、血清学检测法及PCR检测法三类方法的研究进展进行了综述。     

人类微孢子虫检测方法研究进展

微孢子虫(microsporidia)是一类专性细胞内寄生的单细胞真核生物。是引起微孢子虫病的真菌类病原。在已知并被命名的1500多种微孢子虫中,共有9个属中的17个虫种可以感染人。人类微孢子虫可侵染包括肠道、肝、肺、脑等部位,引起慢性腹泻、肝炎、角膜炎、脑炎、血液系统性感染等,严重影响人类健康。研究开发快速高效的人类微孢子虫诊断方法成为当前病原微生物检测领域研究的热点。人类微孢子虫的发现历史实际上是伴随检测方法的不断进步而逐渐进行的。这些检测方法包括,透射电镜(transmission electron microscopy)、苏木精-伊红染色(hematoxylin-eosin stain,HE)、亚甲蓝染色(methylene blue)、吉姆萨染色(giemsa)、革兰氏染色(gram stain)、韦伯氏改良三色染色法(Weber’s chromotrope-based staining)、荧光增白剂染色法(calcofluor white staining)、抗原检测、抗体检测、实时荧光定量PCR (quantitative real-time PCR,q PCR)、环介导...     

O139霍乱弧菌的研究进展

Ｏ１３９霍乱弧菌是一种新发现的可引起霍乱流行的病原体，该病的流行已引起各国的关注。Ｏ１３９霍乱弧菌的研究对菌苗研制和控制疾病有重要意义。本文综述了最近国外Ｏ１３９霍乱弧菌的生物学特性、免疫反应及其毒素共调菌毛（ｔｃｐ）的研究进展。     

霍乱弧菌的致病性与流行性研究进展

综述了近年来霍乱弧菌的致病性和流行性的研究进展,并讨论了流行性和致病性的关系.编码霍乱毒素CT的基因ctxAB并不是霍乱弧菌基因组自身的成分,而是CTXΦ噬菌体基因组的一部分.CTXΦ能特异性地感染并整合至霍乱弧菌的基因组,成为CTX原噬菌体;携带有CTX原噬菌体的霍乱弧菌在环境因素的诱导下,也能向外界分泌CTXΦ噬菌体.RS1基因元件与CTX基因元件在染色体位置上紧邻,它们在功能上也紧密相关,RS1元件能利用CTXΦ的基因形成一个丝状噬菌体RS1Φ,并能向细胞外分泌,进行水平传播;VPI来源于VPIΦ,VPIΦ能在霍乱弧菌的菌株之间传播;VPI是霍乱弧菌获得CTX基因元件的桥梁.最近,又发现了两个与霍乱大流行相关的两个基因区域,VSP-Ⅰ和VSP-Ⅱ.     

应用免疫荧光菌球法快速检测霍乱弧菌的实验研究

1材料与方法1．1实验材料1．1．1菌种E1－Tor生物型霍乱弧菌,菌号103,小川血清型,由军事医学科学院提供。1．1．2免疫荧光血清购于长春生物制品研究所,批号940205。1．1．3培养基Ⅰ号增菌液（碱性胨水）,Ⅱ号增菌液（碱性陈水中加入1：200000亚碲酸钾、1：100000十二烷基磺酸钠）,由本室制备。双氢链霉素洗衣粉亚硫酸钾琼脂培养基（双洗琼脂培养基）,购手上海市卫生防疫站。1．1．4器材①接种环：单环接种环内径为3nun,多环接种环用4个内径3rum环排列成花瓣状,由本室自制。②微量吸引器：由上海生物实验研究所提供。③荧光显微镜…     

DNA环介导恒温扩增技术快速检测霍乱弧菌

霍乱弧菌是一种重要的食源性致病菌,主要引起急性肠道传染病,其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列,设计2对特异性检测引物,利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),经反应体系优化,成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃,60min完成检测,对培养菌的检测限为25CFU/mL,污染食品中霍乱弧菌的检测限为32CFU/g。对33株同种或近源细菌进行LAMP检测,仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明,对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测,共检出85份阳性,与国际标准(ISO TS21872-1-2007)检测结果的符合率为100%。结果表明,本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便,有利于霍乱弧菌疫情的监测。     

PCR检测霍乱弧菌RTX吸毒素基因

建立了一种扩增最近发现的霍乱弧菌RTX毒素基因的PCR方法。在世界各地引起流行和散发霍乱病例的166株临床和环境霍乱弧菌分离物中,用PCR和Hep—2细胞细胞毒性试验发现它们都是产毒的。而在相关基因簇中有缺失的古典生物型标准株用这两种方法结果都是阴性。这是第一个用于鉴别O1群霍乱弧菌古典生物型菌株和E1　Tor生物型菌株以及包括O139血清型的其它非O1群菌株的快速基因分型方法。建立的PCR方法也可特异性地检测霍乱弧菌中的RTX毒素基因,因为用此RTX毒素特异性PCR以及Hep—2细胞毒性试验时副溶血弧菌、致腹泻性大肠杆菌、气单胞菌和邻单胞菌的临床分离株都是阴性。这些结果使霍乱弧菌中RTx毒素的特性明显了。其在细菌致病中的作用需要进一步研究。     

Analysis of some factors involved in the virulence mechanism of Vibrio cholerae non-01

Abstract A study was carried out to evaluate the potential intestinal toxicity of 188 samples of Vibrio cholerae non-01 isolated from seawater found along the beaches of Rio de Janeiro city. Three different assays were carried out involving: (a) detection of vascular permeability factor (PF) in guinea pigs (together with assessment of two culture media for production of the toxin); (b) intestinal fluid accumulation (FA) in suckling mice; and (c) detection of haemolysin. The results demonstrated that both culture media gave a similar level of performance. In the animal assays, 43% of the samples induced PF in guinea pigs, 28.7% caused intestinal fluid accumulation in suckling mice, and 63.28% contained haemolysin. Only 4.25% of the samples gave positive results in all three tests.     

O1群和O139群霍乱弧菌主要抗原研究进展

霍乱弧菌是引起人和动物烈性肠道传染病霍乱的病原体。在霍乱弧菌的200多个血清群中,只有O1群和O139群霍乱弧菌能引起霍乱。快速准确检测O1群和O139群霍乱弧菌是霍乱防治的关键。表面抗原在O1群和O139群霍乱弧菌检测中发挥着重要作用。简要综述了O1群和O139群霍乱弧菌的脂多糖、霍乱肠毒素、外膜蛋白W、毒素共调菌毛和甘露糖敏感血凝素等5种主要抗原的研究进展。     

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.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

霍乱弧菌生物被膜发育与环境调控

生物被膜状态的霍乱弧菌具有极强的环境适应性和超高的感染性,生物被膜的发育调控研究对霍乱弧菌的宿主感染和环境适应非常重要。本文综述了近年来霍乱弧菌生物被膜研究结果,包括霍乱弧菌生物被膜的组成、发育和环境调控,尤其着重阐述了各种环境因子对霍乱弧菌生物被膜发育的影响,包括细菌自体信号分子、自然环境因子和宿主信号分子。     

我国霍乱弧菌的脂肪酸分型研究

目的 对脂肪酸分型方法在霍乱弧菌菌株鉴定、菌株相似性分析等方面的应用价值进行评价。方法 选取了分离自我国的两个主要致病血清群的194株霍乱弧菌菌株（1961年以来的El Tor型和1992年以来的O139群）,提取脂肪酸,应用MIDI公司的脂肪酸分型系统,进行数据分析。结果 检测的所有菌株都含有的脂肪酸成分有13种。霍乱弧菌的判断符合率为88.6%。二维聚类分析没有成明显可区分的群, O139群霍乱弧菌的脂肪酸组成与O1群的相似,产毒与非产毒霍乱弧菌的脂肪酸成分没有显著差异。结论 脂肪酸分型对弧菌种的快速鉴定有应用价值,对霍乱弧菌的现场分离鉴定有辅助意义,在小样本暴发资料的研究中能够反映菌株之间的亲缘关系,但其对霍乱弧菌种属内的各种特征性菌群不具有鉴别能力。     

Localization of DnaK and GroEL in Vibrio cholerae

Though the GroEL and DnaK heat shock proteins are well characterized in prokaryotes, only scanty and controversial information exist about their cellular localization. In the present study, the localization of the heat shock proteins DnaK and GroEL in normal and heat shocked cells of Vibrio cholerae, was investigated both by immunogold labeling of ultrathin sections and biochemical methods. Much of the DnaK was found to be localized at the inner membrane in unstressed cells, most probably at the Bayer's adhesion sites. Data suggested that upon heat shock, the DnaK associated with the membrane continued to remain there, but the newly synthesized DnaK appeared mostly in the cytoplasm. GroEL in both stressed and unstressed cells was found mainly in the cytoplasm.     

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.     

A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis

Vibrio fluvialis strain H-08942 was isolated from an infant aged 6 months who was suffering from cholera-like diarrhea in India. This strain showed the typical multidrug-resistance phenotype of an SXT element. It was resistant to sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm) and streptomycin (Sm), in addition to other antibiotics such as ampicillin (Am), furazolidone (Fz), nalidixic acid (Na), and gentamicin (Gm). The SXT element is a Vibrio cholerae-derived integrative and conjugative element (ICE) that has also been referred to as a conjugative transposon. Our goal was to find a relationship between these resistant phenotypes and the presence of the SXT element in this unique strain. By using PCR, we detected the antibiotic resistance genes, the integrase gene and the attP attachment site of SXT element. Cloning and DNA sequencing results showed that both the SXT integrase gene and its attP site of V. fluvialis were similar but not identical to those of V. cholerae. The SXT integrase gene of V. fluvialis has a 99% identity to that of V. cholerae, and the attP site of SXT of V. fluvialis is variant and shorter (641 bp) than that of V. cholerae (785 bp). It was possible for the SXT of V. fluvialis to be transferred by conjugation to a laboratory strain of Escherichia coli. Here, we report the detection of a variant SXT element in species other than V. cholerae, with molecular characterization and analysis of its integrase gene and its attP site.