霍乱弧菌LAMP快速检测方法的建立及应用

目的 建立环介导等温扩增技术(LAMP)快速检测霍乱弧菌.方法 针对霍乱弧菌外膜蛋白的ompW基因设计特异引物,优化反应条件,建立霍乱弧菌的LAMP检测方法.通过对47株细菌及模拟污染现场,检测该方法的灵敏度、特异度和实用性.结果 活菌计数方法表明建立的LAMP方法检测霍乱弧菌的灵敏度为1.6×10~2 cfu/ml.在人工污染霍乱弧菌的粪便及海水标本中检测的敏感度为1.6×10~3 cfu/ml,在牛奶标本中敏感度为1.6×10~4 cfu/ml.检测21株霍乱弧菌均为阳性,26株非霍乱菌则全部阴性,特异度为100%.结论 建立的LAMP方法检测霍乱弧菌灵敏度、特异度高,可用于霍乱现场监测和流行病学调查.

Abstract：

Objective To establish a loop-mediated isothermal amplification (LAMP) method for rapid diagnosis of Vibrio cholerae. Method Based on the ompW nucleic sequence of Vibrio cholerae, a pair of primers was designed for LAMP. The reaction conditions were optimized, and the specificity, sensitivity, and practicability of LAMP were tested using 47 baterial strains and simulated contaminated sites. Results The results of viable bacterium count showed that LAMP was capable of detecting Vibrio cholerae at a level as low as 1.6×10~2 cfu/ml. The minimal detectable concentration was 1.6×10~3 cfu/ml for simulated contaminated samples such as feces and seawater, and 1.6×10~4 cfu/ml for contaminated milk. All the 21 strains of Vibrio cholerae yielded positive results in LAMP, and the 26 strains of other bacteria all showed negative results, with a detection specificity of 100%. Conclusion The established LAMP method has high specificity and sensitivity for detecting Vibrio cholerae and is applicable in field monitoring and epidemiological study of Vibrio cholerae.     

改良分子信标-实时PCR快速检测霍乱弧菌

目的：建立改良分子信标-实时PCR检测霍乱弧菌的快速方法，应用于霍乱监测。方法：根据GenBank公布的霍乱弧菌肠毒素基因A亚单位(ctxA)的保守序列，设计一对引物和改良分子信标探针，并进行特异性和灵敏度分析；同时以11种细菌作对照，建立改良分子信标检测霍乱弧菌的实时PCR反应体系，应用于霍乱监测。结果：霍乱弧菌改良分子信标检测体系检测12种细菌，只有霍乱弧菌有荧光信号，与其他细菌无交叉反应，DNA灵敏度为102．4fg／ul，菌液灵敏度为32cfu／ml或3cfu／PCR反应体系。对100份海产品和30份腹泻病人大便标本进行检测，结果都为阴性。检测时间仅需2h。结论:改良分子信标-实时PCR检测体系快速、灵敏度高、特异性强，可用于霍乱的快速诊断，为霍乱的分子流行病学调查提供新的检测手段。     

ICU多重耐药鲍曼不动杆菌的耐药性和同源性分析

目的通过对重症监护病房（ICU）的多重耐药鲍曼不动杆菌进行同源性分析,进行院内感染的基线调查,并分析其耐药谱特征,为临床防治和控制院内感染提供依据。方法测定17种抗菌药物对62株鲍曼不动杆菌的最低抑菌浓度（MIC）,并采用随机扩增多态性DNA（RAPD）技术分析其耐药株的同源性。结果有16株鲍曼不动杆菌对头孢菌素类、氨基甙类和氟喹若酮类等抗菌素均显示出较高水平的多重耐药性;其中4株RAPD分型显示有高度同源性。结论分离自ICU的鲍曼不动杆菌多重耐药率高达21％,且大多数菌株有高度同源性。因此建立全面的药敏谱和RAPD基因分型体系,加强鲍曼不动杆菌耐药监测,为及时找到感染源头、有效控制医院感染提供依据。     

多重耐药鲍曼不动杆菌的耐药性和同源性研究

目的 了解重症监护病房(ICU)的多重耐药鲍曼不动杆菌的耐药谱特征及其同源性,为临床防治和控制院内感染提供依据.方法 收集该院重症监护病房2007年3～9月分离出的14株鲍曼不动杆菌,采用E-test法测定15种抗茵药物的最低抑菌浓度(MIC)并采用随机扩增多态性DNA(RAPD)技术分析其耐药株的同源性.结果 14株茵对头孢菌素类,氨基甙类和氟喹若酮类等抗菌素均显示出较高水平的多重耐药性;RAPD分型11株菌有高度同源性.结论 分离自ICU的鲍曼不动杆菌多重耐药率较高,且大多数菌株有高度同源性.     

甲氧西林耐药溶血葡萄球菌的耐药基因检测及同源性分析

目的 了解本地区甲氧西林耐药溶血葡萄球菌(MRSH)携带耐药基因情况及其流行状况,提供临床治疗及感染控制的依据.方法 耐药基因的检测采用PCR,菌株同源性分型采用随机扩增DNA 多态性(RAPD)技术.结果 65 株MRSH 的mecA、Aac(6')/aph(2')、aph(3')-Ⅲ、ermB、ermC 和tetM 耐药基因的阳性率分别为100.0%、93.8%、76.9%、7.7%、96.9%和4.6%,未检测出ermA、tetO 和vanA 耐药基因.依照RAPD 检测方法将65 株MRSH 分为3型,其中A 型占83.1%.结论 本地区的MRSH 携带高比例的mecA、Aac(6')/aph(2')、aph(3')- Ⅲ和ermC 耐药基因,RAPD分析MRSH 的流行株以A 型为主.     

霍乱弧菌毒力表达调控基因toxR的PCR扩增及克隆

为了构建霍乱弧菌和杜氏利什曼原虫双价口服活疫苗候选株,作者采用PCR对霍乱弧菌毒力表达调控基因toxR进行扩增及克隆,并对霍乱弧菌toxR基因进行限制性酶切分析.结果显示:7株霍乱弧菌均扩增出1.3kb的toxR基因片段,toxR基因中部含有EcoRⅠ酶切位点,再将toxR基因克隆在质粒pAT153上,对所获的重组质粒ptR4进行限制性酶切分析,证实质粒ptR4插入的1.3kb的DNA片段为toxR基因片段.     

The inherent characteristics and DNA polymorphism of Vibrio cholerae and other vibrios

Objective To investigate the inherent characteristics of Vibrio cholerae (V.cholerae) and other vibrios and their relationship.Methods Polymerase chain reaction (PCR), DNA sequence analysis, randomly amplified polymorphic DNA (RAPD) analysis and average linkage cluster analysis were used to study 3 isolates of V.cholerae strains O139, three isolates O1 biot ype El Tor, four isolates O1 biotype classical and 3 other vibrios.Results V.cholerae O139 contained the genomic sequences of ctx A2- B as well as V.cholerae O1.V.cholerae and others vibrios were divided into 4 groups by fingerprint patterns of RAPD, that is ① V.cholerae O139 and V.cholerae O1 El Tor; ② V.cholerae O1 classical; ③ V.para heamolyticus and V.vulnificus and ④ V.flluvialis.V.cholerae O139 DNA fingerprint of RAPD was consistent with the El Tor biotype: average linkage cluster distance was 0, and slightly different from the classical b iotype, with a distance of 2.07.It was much more different from vibrio par aheamolyticus and others, with a distance of 6.76-8.54Conclusion V.cholerae and other vibrios are polymorphic in inherent characteristics. The inherent characteristics of V.cholerae O139 are the same as El Tor b iotype.O139 may have evolved from the El Tor biotype.The inherent char acteristics of vibrio paraheamolyticus are the same as vibrio vulnificus.     

小鼠RAPD-PCR反应体系及扩增程序的筛选

目的:筛选优化小鼠RAPD-PCR反应体系及扩增程序.方法:以40条引物对9个品系的小鼠基因组DNA在各种不同条件下进行RAPD-PCR,分析比较扩增结果.结果:在下列反应体系及扩增程序下结果较好:在25 μl的反应体系中含有10×buffer 2.5 μl,dNTP 200μmol/L, Mg2+ 2.5 mmol/L,引物0.2 μmol/L,Taq酶1.5 U,模板DNA 20 ng,扩增程序为94℃变性1 min,38℃退火1 min,72℃延伸2 min,40个循环后接10 min延伸.结论:以上述反应体系及扩增程序进行小鼠RAPD-PCR,扩增结果稳定.     

BALB/c突变无毛小鼠特异性免疫功能的研究

选用9条随机引物，对Wistar,WKY,F344三个品系的近交系大鼠进行了随机扩增多态性DNA（RAPD）分析。结果显示同一品系大鼠不同个体之间的RAPD图谱都相同，表明所测的三个品系大鼠内部遗传背景均一，在不同品系大鼠之间的RAPD结果有差异。Wistar与WKY之间，Wistar与F344之间，WKY与F344之间的共享度分别为87.9%,98.5%和89.2%。RAPD方法可以作为一种检测近交系大鼠种群内的遗传变异及区分不同品系近交系鼠的遗传检测方法。     

O1群和O139群霍乱弧菌荧光PCR快速检测方法的建立

目的建立检测O1群和O139群霍乱弧菌的实时荧光聚合酶链反应（荧光PCR）方法,并进行优化和评价。方法根据O1群和O139群霍乱孤菌O抗原编码基因设计探针和引物,建立同时检测霍乱孤菌O1群和O139群的荧光PCR方法,并对体系中的引物、Mg^2＋、dNTP和Taq酶进行优化,然后对建立的方法进行特异性、灵敏性、重复性的评价,并进行224份河口水样本的检测。结果建立了检测O1群和O139群霍乱弧菌的双重荧光PCR方法。对非O1群和O139群霍乱孤菌无扩增反应,敏感度比常规分离培养高。结论以O抗原编码基因为目标检测片段建立了O1群和O139群霍乱孤菌双重荧光PCR检测方法,可用于疑似霍乱弧菌感染的样本常规分离前的快速筛查。     

LAMP方法检测霍乱弧菌的研究

目的建立一种快速检测霍乱弧菌的方法。方法利用霍乱弧菌的hlyA基因设计特异性引物,建立LAMP检测方法,以及优化反应体系,并进行LAMP的特异性和灵敏度实验,同时与普通PCR和荧光定量PCR进行比较。结果成功建立霍乱弧菌的LAMP检测方法,得到最优的反应体系,在特异性实验中,霍乱弧菌均呈阳性,而副溶血弧菌、溶藻孤菌、最小孤菌、1梅氏孤菌等均为阴性;在灵敏度实验中,霍乱弧菌的最低检测限为6.2pg／tube,是普通PCR的1000倍。比荧光定量PCR低一个数量级。结论LAMP方法具有很高的特异性和灵敏度,可用于霍乱孤菌的检测。     

霍乱肠毒素基因DNA疫苗的构建和体外表达

目的构建霍乱弧菌ctxAB融合基因分子佐剂DNA疫苗,并在NIH3T3细胞中进行表达。方法用限制性核酸内切酶从原核表达重组质粒pET32a-ctxAB上切下ctxAB基因,导入真核表达载体pcDNA3.1（＋）,重组子经限制性酶切分析、PCR鉴定正确后,命名为pcDNA3.1-ctxAB。用脂质体法将重组质粒pcDNA3.1-ctxAB转染NIH3T3细胞,采用免疫荧光法和Western blot对pcDNA3.1-ctxAB的瞬时表达产物和稳定表达产物进行鉴定。结果真核表达重组质粒pcDNA3.1-ctxAB成功转入NIH3T3细胞,利用免疫荧光技术检测到其在细胞膜和细胞浆中获得瞬时表达,对稳定转染细胞用Western blot分析,发现在约40.5kDa处有阳性杂交信号。结论成功构建霍乱弧菌ctxAB基因分子佐剂DNA疫苗,并在NIH3T3细胞中获得表达。     

霍乱弧菌O139和El Tor菌株毒力岛区域分子特征比较

目的:比较O139霍乱弧菌(VC O139)菌株MO45基因组与O1群El Tor霍乱弧菌(EVC)菌株N16961基因组中毒力岛(VPI)区域分子特征.方法:制备霍乱弧菌VPI上游VC0815基因探针.从VC O139菌株MO45基因组文库中筛选一个既含有VC0815基因又含有tcpA基因的克隆,命名为pCOSVC081545,绘制pCOSVC081545 VPI上游片段的限制性酶切图谱,并与N16961同段序列的限制性酶切图谱进行比较.结果与结论:pCOSVC081545和N16961中VPI上游约1 500 bp片段的限制性酶切图谱基本相同,支持O139霍乱弧菌是EVC O抗原突变而来的观点.     

霍乱弧菌HutX蛋白的晶体结构

目的 获取纯化的霍乱弧菌HutX蛋白晶体和衍射数据.方法 通过基因克隆和蛋白表达得到HutX蛋白,通过镍柱亲和层析、Source Q阴离子交换柱层析和Superdex 200分子筛柱层析等纯化所得蛋白,并用蛋白质印迹法进行验证.将所获蛋白进行结晶条件筛选及悬滴优化,X线衍射分析所得晶体的结构.结果 蛋白质印迹分析间接证明所得到的蛋白为HutX蛋白,并得到HutX蛋白的晶体及衍射数据.结论 为进一步研究HutX的蛋白结构及其在霍乱弧菌亚铁血红素利用机制中发挥的作用提供了参考.     

Toxin-coregulated pilus-loaded microparticles as a vaccine against Vibrio cholerae O139

The cholera epidemics is an important public health problem in many developing countries. Highly effective and preventive vaccines against cholera are under investigation as alternatives to the one available presently. Much of the vaccine research focuses on colonization factors. Colonization of a human by the Vibrio cholerae (V. cholerae Ol strain is mediated by toxin-coregulated pilus (TCP), 1 which was shown to play a role in the infant mouse cholera model and subsequently in human volunteers. 2 TCP-loaded vaccines could potentially provide cross-protection among experimental strains. Data have indicated that poly (D,L-lactide)-polyethylene glycol copolymer (PELA)microparticles loaded antigens were strongly immunogenic, 3 and that these microparticles served as an effective delivery system for a single dose of vaccine. 4Microparticle formulation could represent the next generation of vaccines, as they are highly effective at delivery of vaccines, thus requiring fewer doses. 5 We prepared PELA microparticles loaded with TCP for testing as a vaccine; their targeting distributions were identified and related immune responses were analyzed.     

Toxin-coregulated pilus-loaded microparticles as a vaccine against Vibrio cholerae O139

The cholera epidemics is an important public health problem in many developing countries. Highly effective and preventive vaccines against cholera are under investigation as alternatives to the one available presently. Much of the vaccine research focuses on colonization factors. Colonization of a human by the     

霍乱弧菌O1群菌株的群体遗传学特征

霍乱的病原菌是Ｏ１群霍乱弧菌，但在Ｏ１群霍乱弧菌中，仅有部分可以引起霍乱流行，因此不同菌株在流行病学上的意义差异很大。为探讨Ｏ１群菌不同型别和亚群之间的遗传关系和进化变异规律，应用多位点酶电泳法对Ｏ１群霍乱弧菌进行研究。结果：Ｏ１群霍乱弧菌的大多基因位点等位基因频率较低，尤其是古典型菌株和埃尔托型流行株，大多被检测基因为非多态位点；Ｏ１群霍乱弧菌的平均遗传多态值（ＨＯ１）为０．１８３，平均杂合度（ｈＯ１）为５．６７；其中古典型菌、埃尔托型流行株、埃尔托型非流行株的平均遗传多态值和平均杂合度分别为：ＨＣＶＣ＝０．１０６，ｈＣＶＣ＝１．５；ＨＥＳＥＶＣ＝０．０５６，ｈＥＳＥＶＣ＝２．３；ＨＮＳＥＶＣ＝０．４２５，ｈＮＳＥＶＣ＝５。结果显示：古典型和埃尔托型流行株作为霍乱的病原体是一个遗传关系紧密的群体，而埃尔托型非流行株遗传关系较为离散，与流行株之间的关系也较远，可能是其长期作为环境菌株进化变异的结果     

Structural variation of the superintegron in the toxigenic Vibrio cholerae O1 El Tor

Objective To understand the genetic structures and variations of the superintegron(SI)in Vibrio cholerae isolated in the seventh cholera pandemic.Methods Polymerase chain reaction scanning and fragment sequencing were used.Sixty toxigenic V.cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed.Results Some variations were found,including insertions,replacements,and deletions.Most of the deletions were probably the result of recombination between V.cholerae repeat sequences.The majority of ...     

霍乱弧菌不同群体菌株的群体遗传特征比较

应用ＭＥＥ法对５９２株霍乱弧菌的１５个酶基因位点进行检测，分析和比较霍乱弧菌不同群体的群体遗传特征。结果：古典型菌株（ＣＶＣ）、埃尔托流行株（ＥＳＥＶＣ）和Ｏ１３９菌株（Ｏ１３９）的群体平均遗传多态值和平均杂合度都很低，ＨＣＶＣ＝０．１０６、ｈＣＶＣ＝１．５，ＨＥＳＥＶＣ＝０．０５６、ｈＥＳＥＶＣ＝２．３（Ｏ１３９包含在ＥＳＥＶＣ之中），被检测的基因位点多为非多态位点；而同为Ｏ１群菌的埃尔托非流行株（ＮＳＥＶＣ）的平均遗传多态值和平均杂合度要高得多，接近非Ｏ１群菌株（ＮＯ１）的群体遗传学指标，分别为ＨＮＳＥＶＣ＝０．４２５、ｈＮＳＥＶＣ＝５，ＨＮＯ１＝０．６２７、ｈＮＯ１＝６．５，被检测的基因位点几乎全是遗传多态位点。结果表明：ＥＳＥＶＣ和ＮＳＥＶＣ虽然都是Ｏ１群菌，但在群体遗传学特征上存在巨大差异；ＣＶＣ、ＥＳＥＶＣ和Ｏ１３９为遗传学性状稳定、群体遗传结构紧密的群体，而ＮＳＥＶＣ和ＮｏｎＯ１的群体遗传关系离散