研究由新罕布什尔州INBRE通过机构发展奖（IDeA）支持，P20GM103506来自美国国家卫生研究院国家综合医学研究所。

注意:每个测定的时间考虑必须作为单个培养基制备需要不同的时间。例如，应将固体琼脂平板培养基充分冷却并干燥（1-2天）。额外的时间考虑（ 即单菌落和隔夜培养生长）在每个方案下规定， 见表2 。 媒体准备<ol><li> 1x磷酸缓冲盐水（PBS） <ol><li>称取4.0g NaCl，0.1g KCl，0.72g Na 2 HPO 4和0.12g KH 2 PO 4 。将组分在1升锥形烧瓶中，用去离子水将体积调节至500mL，使用pH计将pH调节至7.2，并将HCl滴加到溶液中，并使用0.22μm过滤器进行高压灭菌或过滤灭菌。 1x PBS可以无限期地在室温下储存。 注意:HCl是浓缩酸，应按照以下方式处理到制度，地方，州和联邦法规。对于适当的处理指南，请参阅制造商提供的安全数据表。 </li></ol></li><li> Luria-Bertani（LB）肉汤 <ol><li>称重5.0g胰蛋白胨，2.5g酵母提取物和2.5g NaCl。将组分在1升瓶中，用去离子水调节体积至500mL，高压灭菌，并在室温下储存长达6个月。对于经典的生物型毒力诱导条件，在高压灭菌前用HCl调节pH至6.5。 </li></ol></li><li> 含有0.03％（w / v）NaHCO 3的 AKI培养基 <ol><li>对于组分1（AKI培养基）:重7.5g蛋白胨，2.0g酵母提取物和2.5g NaCl。将组分在1升瓶中，用去离子水和高压釜将体积调节至450mL。 AKI培养基可以在室温下储存长达6个月。 </li><li>对于组分2（NaHCO 3 ）:称重1.5g NaHCO 3 ，并调节体积在无菌囊泡中用去离子水加至50mL。使用0.22μm过滤器过滤灭菌NaHCO 3 。必须在使用前立即制备NaHCO 3 。 </li><li>在使用前将组分2过滤到组分1中，无菌地组合两种成分，产生1:10的比例。 </li></ol></li><li> Luria-Bertani（LB）琼脂板 <ol><li>称取5.0g胰蛋白胨，2.5g酵母提取物，2.5g NaCl和7.5g琼脂。将组分在1升锥形烧瓶中，用去离子水，高压釜调节体积至500毫升，并在标准尺寸的100毫米×15毫米无菌培养皿中制备。电镀介质可以保存在塑料中长达6个月，盖子朝上放置在4°C。 </li></ol></li><li> LB琼脂平板补充多粘菌素B <ol><li>对于组分1（LB琼脂）:称重5.0g胰蛋白胨，2.5g酵母提取物，2.5g NaCl和7.5g琼脂。将组分在1升锥形瓶中，将体积调节至500mL wi去离子水和高压釜。 </li><li>对于组分2（多粘菌素B）:将无菌2mL微量离心管中的去离子水中的多粘菌素B溶解至50,000IU /μL的浓度，并使用0.22μm过滤器进行过滤消毒。 </li><li>一旦组分1冷却，但尚未固化，无菌地向组分1中加入500μL组分2，产生最终浓度为50IU /μL并充分混合。通过将混合的琼脂培养基倒入培养皿中制备标准尺寸的100mm×15mm无菌培养皿。电镀介质可以保存在塑料中长达3个月，盖子在4℃升高。 </li></ol></li><li> 柠檬酸盐中等琼脂平板 <ol><li>对于组分1（具有溴百里酚蓝的琼脂）:称重7.5g琼脂和0.02g溴百里酚蓝。将组分在1升三角烧瓶中合并，用去离子水将体积调节至450mL，并将混合物高压。 </li><li>对于组件2（10x VBMM）:称重10g MgSO 4 7H 2 O，10.0g柠檬酸H 2 O，50.0g无水K 2 HPO 4和17.5g NaNH 4 HPO 4 4H 2 O。将组分在1升锥形瓶中，将体积调节至500mL用去离子水和高压灭菌器或使用0.22μm过滤器进行过滤消毒。 10x VBMM可在室温下储存6个月。 </li><li>通过将混合的琼脂培养基倒入培养皿中，将50 mL组分2加入到组分1中，并制成标准尺寸的100 mm x 15 mm无菌培养皿。电镀介质可以保存在塑料中长达6个月，盖子朝上放置在4°C。 </li></ol></li><li> 牛奶琼脂板 <ol><li>对于组分1（牛奶）:在1L三角烧瓶中称重8.0g速溶脱脂乳，用去离子水和高压釜调节体积至200mL。 </li><li>对于组分2（具有脑心脏输注的琼脂）:称重3.68g脑 - 心脏输注和6.0g琼脂。结合con在500mL三角烧瓶中的成分，用去离子水和高压釜将体积调节至200mL。 </li><li>通过将组分2倒入组分1中，高压灭菌后彻底混合，合并两种组分。在150毫米x 15毫米无菌培养皿中准备。牛奶板可以储存长达一周，塑料包装，盖子侧面在4°C下方。通过将混合的琼脂培养基倒入培养皿中制备150毫米×15毫米无菌培养皿。 </li></ol></li><li> 动力琼脂板 <ol><li>对于组分1（LB琼脂）:称重5.0g胰蛋白胨，2.5g酵母提取物，2.5g NaCl和2.0g琼脂。将组分在1升锥形瓶中合并，用去离子水和高压釜调节体积至500mL。 </li><li>对于组分2（1％（w / v）三苯基四唑氯化物; TTC）:称重0.25g TTC。在无菌囊泡中用去离子水将体积调节至25 mL，并使用0.22μm过滤器进行过滤消毒。存放长达6个月的室温包裹在箔中以最小化曝光。 </li><li>加入2.5 mL 1％（w / v）无菌TTC至高压灭菌培养基。 </li><li>将介质尽可能厚（≥50mL /平板）倒入大型150 mm x 15 mm无菌培养皿中。电镀介质可以保存在塑料中长达一周，盖子在4℃升高。通过将混合的琼脂培养基倒入培养皿中制备150毫米×15毫米无菌培养皿。 </li></ol></li><li> Voges-Proskauer（副总裁） <ol><li>对于组分1（甲基红Voges-Proskauer肉汤; MR-VP）:在500mL锥形瓶中重1.7g MR-VP培养基，用去离子水和高压釜调节体积至100mL。无菌MR-VP肉汤可以在室温下储存长达6个月。 </li><li>对于组分2（5％（w / v）α（α）萘酚）:在无菌囊泡中称重1.0gα-萘酚，并用95％乙醇调节体积至20mL。 α-萘酚可以在室温下储存长达1周的箔包裹，以使光透过率最小化osure。 </li><li>对于组分3（40％（w / v）氢氧化钾; KOH）:在无菌泡囊中重量为20.0g KOH，用无菌去离子水将体积调节至50mL。 KOH可以在室温下在塑料容器中储存多达6个月。 </li></ol></li></ol>维持和增长V。 霍乱弧菌菌株<ol><li> 冷冻库存培养的准备和使用 <ol><li>颗粒将1.8mL液体过夜培养2分钟，在无菌2mL微量离心管中离心（≥8,600xg），除去上清液，并通过移液将细胞沉淀重新悬浮在900μL新鲜LB肉汤中。 </li><li>向培养基中加入900μL无菌60％（v / v）甘油，并通过涡旋混合。 </li><li>将混合物转移到无菌2 mL低温管中，无限期储存在-80°C。 </li><li>为了使用，使用无菌接种环和少量冷冻原料，在LB ag上用于单个菌落ar板。使用后立即将冷藏库存回到-80°C，以防止文化完全解冻。将盖板盖在37℃下孵育12至16小时。 </li></ol></li><li> 液体培养基中过夜培养物的生长 <ol><li>将单个菌落（根据2.1.4节）从冷冻原料连续到LB琼脂平板上。将盖板盖在37℃下孵育12至16小时。 </li><li>通过用无菌涂抹棒接触单个菌落的表面，将4mL液体LB肉汤接种在具有单个菌落的无菌10mL培养管中，并将菌落转移到液体肉汤中。 </li><li>在振荡器培养箱中以225rpm的通气温度在37℃下孵育12至16小时。 </li></ol></li><li> 增长曲线 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养。 </li><li>通过将250μL过夜培养物转移到2，进行1:100稀释的过夜培养将5mL新鲜LB肉汤在无菌的250mL锥形瓶中。 </li><li>在接种时开始每小时测量600nm（OD 600 ）的液体培养物的光密度（T 0 ）。 </li><li>孵育1:100稀释的过夜培养物，在振荡器培养箱中以225rpm通气37℃培养达30小时，或培养达到最大密度。 </li><li>将OD 600与时间对照，并使用线性回归确定每个菌株的近似加倍时间。 </li></ol></li><li> El Tor生物型毒力诱导条件 <ol><li>将单个菌落从冷冻原液连续到LB琼脂平板上。将盖子盖板向下孵育，在37℃下放置12至16小时。 </li><li>接种含有0.03％（w / v）NaHCO 3的 10mL含有单个菌落的AKI培养基。 </li><li>孵育培养物，37℃不通气3.5小时。 </li><li>取出7 mL培养液，孵育剩余的3 mL培养液在振荡器培养箱中以225rpm曝气另外4小时。 </li></ol></li><li> 古典生物型毒力诱导条件 <ol><li>将单个菌落从冷冻原液连续到LB琼脂平板上。将盖板盖在37℃下孵育12至16小时。 </li><li>用单个菌落接种4mL液体LB肉汤（pH 6.5）。 </li><li>在振荡器培养箱中以225rpm在30℃下通气曝气12至16小时。 </li></ol></li><li> 在1x PBS中洗涤细胞颗粒 <ol><li>在无菌的2mL微量离心管中通过离心（≥8,600xg）将1.8mL过夜培养物沉淀2mL，并用移液管除去上清液。通过移液将细胞沉淀重悬于1.8 mL 1x PBS。 </li><li>重复洗涤程序三次，最后重悬于1.8 mL 1x PBS中。 </li></ol></li></ol>表征V。 霍乱生物型<ol><li> P使用ctxB和tcpA的基于CR的遗传筛选<ol><li>设计一组引物，分别在ctxB和tcpA的翻译启动和停止位点的上游和下游退火约50-70bp。 </li><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养。 </li><li>使用为革兰氏阴性菌设计的市售试剂盒分离染色体DNA。纯化的染色体DNA可以无限期储存在-20°C。使用市售试剂盒的染色体DNA分离通常需要约4小时。 </li><li>使用微量分光光度计确保染色体DNA样品质量高（A 260/280 &gt; 1.8）。 </li><li>对于每个染色体DNA分离物，在无菌200μLPCR管中在冰上制备聚合酶链式反应（PCR），并使用标准PCR组分扩增ctxB和tcpA的区域: Taq聚合酶和buffer（或等同物），dNTP溶液和正向/反向引物。使用标准PCR参数（〜3 h）。例如，使用以下协议: <ol><li> 95°C初始变性120 s。 </li><li>在95℃变性60秒。 </li><li>退火底漆在60℃45秒。 </li><li>在72℃延伸90秒。 </li><li>重复步骤3.1.5.2至3.1.5.4进行34个周期。 </li><li>最终延伸72°C，持续600 s。 </li><li>无限保持在4°C。 </li></ol></li><li>使用标准的6x DNA凝胶加载染料染色5μL各自的PCR产物，并将大约10μL的1kb梯度和10μLPCR产物加载到1×Tris-硼酸盐-EDTA（TBE）中的1％琼脂糖凝胶上，缓冲。 </li><li>在130 V下运行凝胶电泳，直到染料前端达到凝胶的末端，但不能脱离凝胶（约90分钟）。建议进行恒定监控，因为电压和运行时间因设备而异。 </li><li>经验证以预期的大小成功进行PCR扩增，使用市售的DNA清洁和浓缩器试剂盒纯化剩余的45μLPCR产物。 </li><li>使用与PCR扩增相同的引物进行序列清洗的PCR产物，并根据每个本地测序设备指南进行准备。 </li><li>通过在搜索查询框中搜索以下登录号，将NCAPI网站（http://www.ncbi.nlm.nih.gov/gene/）上可获得的每个分离株的基因序列的FASTA格式与已公布的序列进行比较。 </li><li> ctxB :VC0395_A1059到VC0395_A1060（El Tor）VC_1456之间的（经典）区域</li><li> tcpA（经典）VC0395_A0353（El Tor）VC_0828 </li></ol></li><li> 通过斑点生物型分类的表型分析 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养。 </li><li>洗涤方案2.6中规定的细胞沉淀。 </li><li>使用移液器在各自的培养基上洗涤1μL洗涤的培养物。有关中等选择和孵化规格，请参见表2。 </li></ol></li><li> 动力测定 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养物。 </li><li>洗涤方案2.6中规定的细胞沉淀。 </li><li>通过将接种刺入插入洗涤的液体培养物中并"垂直地"插入培养基中来接种动力琼脂平板。在每次接种之间，使用本生灯灭菌线刺，并且当刺入琼脂时，确保接种刺不弯曲，因为这样可以改变结果。 </li><li>将盖板盖在37℃下孵育14至24小时。 14小时后，密切监测动力板，以防止文化过度生长。 </li></ol></li><li> Voges-Proskauer（VP）测定 <ol><li>如方案2.2中所述，在液体LB肉汤中制备过夜培养物。 </li><li>接种4mL的甲基红Voges-Proskauer或MR-VP肉汤，通过在无菌培养管中将10μL先前制备的过夜培养液移液到4mL MR-VP肉汤中，并在振荡器培养箱中以225rpm通气12至16℃ h在37°C。 </li><li>在无菌培养管中分别加入150μL5％（w / v）α-萘酚和50μL40％（w / v）KOH至1mL等分的MR-VP过夜培养。 </li><li>短暂旋转并在室温下放置4小时，直到发生变色。 </li></ol></li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+spread+of+cholera:+genetic+diversity+and+relationships+within+the+seventh+pandemic+clone+of+Vibrio+cholerae+determined+by+amplified+fragment+length+polymorphism.">Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism.</a> J. Clin. Microbiol. 40 (1), 172-181 (2002).</li><li>Nair, G. B., Faruque, S. M., Bhuiyan, N. A., Kamruzzaman, M., Siddique, A. K., Sack, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+variants+of+Vibrio+cholerae+O1+biotype+El+Tor+with+attributes+of+the+classical+biotype+from+hospitalized+patients+with+acute+diarrhea+in+Bangladesh.">New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh.</a> J. Clin. Microbiol. 40 (9), 3296-3299 (2002).</li><li>Nair, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Vibrio+cholerae+O1+strains+similar+to+pre-seventh+pandemic+El+Tor+strains+during+an+outbreak+of+gastrointestinal+disease+in+an+island+resort+in+Fiji.">Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji.</a> J. Med. Microbiol. 55 (11), 1559-1562 (2006).</li><li>Nair, G. B., Mukhopadhyay, A. K., Safa, A., Takeda, Y., Faruque, S. M., Nair, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+hybrid+variants+of+Vibrio+cholerae+O1.">Emerging hybrid variants of Vibrio cholerae O1.</a> Vibrio cholerae—Genomics and Molecular Biology. , 179-190 (2008).</li><li>Safa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+characteristics+of+Matlab+variants+of+Vibrio+cholerae+O1+that+are+hybrids+between+classical+and+El+Tor+biotypes.">Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes.</a> J. Med. Microbiol. 55 (11), 1563-1569 (2006).</li><li>Nusrin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+CTX+phages+among+toxigenic+Vibrio+cholerae+O1+and+O139+strains+isolated+between+1994+and+2002+in+an+area+where+cholera+is+endemic.">Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic.</a> J. Clin. Microbiol. 42 (12), 5854-5856 (2004).</li><li>Carignan, B. M., Brumfield, K. D., Son, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphisms+in+regulator-encoding+genes+have+an+additive+effect+on+virulence+gene+expression+in+a+Vibrio+cholerae+clinical+isolate.">Single nucleotide polymorphisms in regulator-encoding genes have an additive effect on virulence gene expression in a Vibrio cholerae clinical isolate.</a> mSphere. 1 (5), e00253 (2016).</li><li>Iwanaga, M., Yamamoto, K., Higa, N., Ichinose, Y., Nakasone, N., Tanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+stimulating+cholera+toxin+production+by+Vibrio+cholerae+O1+El+Tor.">Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor.</a> Microbiol. Immunol. 30 (11), 1075-1083 (1986).</li><li>DiRita, V. J., Claude, P., Georg, J., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+cascade+controls+virulence+in+Vibrio+cholerae.">Regulatory cascade controls virulence in Vibrio cholerae.</a> Proc. Natl. Acad. Sci. USA. 88 (12), 5403-5407 (1991).</li><li>Kovacikova, G., Lin, W., Skorupski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duel+regulation+of+genes+involved+in+acetoin+biosynthesis+and+motility/biofilm+formation+by+the+virulence+activator+AphA+and+the+acetate-responsive+Lys-R+type+regulator+AlsR+in+Vibrio+cholerae.">Duel regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive Lys-R type regulator AlsR in Vibrio cholerae.</a> Mol. Microbiol. 57 (2), 420-433 (2005).</li><li>Vogel, H. J., Bonner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylornithinase+of+Escherechia+coli:+partial+purification+and+some+properties.">Acetylornithinase of Escherechia coli: partial purification and some properties.</a> J. Biol. Chem. 218 (1), 97-106 (1956).</li><li>Son, M. S., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+and+biochemical+assays+to+characterize+Vibrio+cholerae+O1+biotypes:+classical+and+El+Tor.">Genetic screens and biochemical assays to characterize Vibrio cholerae O1 biotypes: classical and El Tor.</a> Curr. Protoc. Microbiol. , 6A.2.1-6A.2.17 (2011).</li><li>Kovacikova, G., Skorupski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+virulence+gene+expression+in+Vibrio+cholerae+by+quorum+sensing:+HapR+functions+at+the+aphA+promoter.">Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter.</a> Mol. Microbiol. 46 (4), 1135-1147 (2002).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+functional+quorum-sensing+systems+in+recently+emerged+Vibrio+cholerae+toxigenic+strains.">The prevalence of functional quorum-sensing systems in recently emerged Vibrio cholerae toxigenic strains.</a> Environ. Microbiol. Rep. 3 (2), 218-222 (2011).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques.">Aseptic laboratory techniques.</a> J. Vis. Exp. (63), e3064 (2012).</li><li>Martinez, R. M., Megli, C. J., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+laboratory+maintenance+of+Vibrio+cholerae.">Growth and laboratory maintenance of Vibrio cholerae.</a> Curr. Protoc. , 6A.1.1-6A.1.6 (2010).</li></ol>

作者没有什么可以披露的。

在200多个确定的V。 霍乱血清群，只有O1和O139具有流行潜力。 O1血清群可以分为两种生物型:古典和El Tor。然而，已经出现了称为El Tor变体13,17的杂交菌株，具有El Tor生物型背景，并具有古典特征8,9,10,11,12,13,14,15,16,17。本手册中描述的方案旨在提供有兴趣表征和/或区分霍乱弧菌的各种临床和非临床分离株的研究者，具有可靠的多检测鉴定系统。使用可靠的多分析鉴定系统作为替?...

霍乱是由食用含有水生革兰氏阴性菌霍乱弧菌的污染食物或水引起的远端小肠疾病。 霍乱的症状包括呕吐和不可控制的水样腹泻，导致严重脱水，如果不妥善治疗，将导致死亡。 V。基于细胞表面脂多糖O-抗原的结构， 霍乱弧菌可以分为200多个血清群。然而，只有2个血清群，O1和O139，已显示出流行或大流行的潜力1,2 。此外，血清群O139主要分离到东南亚3,4 ，而血清群O1在全球分布。此外，O1血清群可以分为两种主要生物型:古典和El Tor。经典生物型是造成头6头霍乱流行病1817年和1923年之间。正在进行的第七大流行病是El Tor生物型的结果，全球在环境5,6,7的环境中使古典生物型流行病学变异。最近，出现了具有古典和El Tor生物型8,​​9,10,11,12,13,14,15,16,17的区别特征的菌株，并且之后被称为El Tor变体13,17。一些El Tor变体已经显示出比以前观察到的更迅速和严重的疾病进展的升高的毒力能力，强调需要一个更全面的方法来代理身份识别和疾病预防和治疗8,9,18。虽然生物型鉴定并不立即决定治疗，但疫苗开发和未来治疗剂的进一步进步可能受益于生物型别的区别。 这里列出的第一系列协议将使调查人员能够正确维护V。 霍乱弧菌菌株在实验室设置。一致性和随后的分析需要分离物的储备准备和生长，其不是生物型依赖性的。然而，为了最佳地诱导毒力基因表达，需要独立的生物特异性培养技术。此外，本手稿中概述了各种遗传和生物化学测定的准备工作。 霍乱毒素（CT）和毒素共同作用培养的真菌（TCP）是主要调节剂ToxT在V的两种生物型中控制的两种主要的毒力因子。 霍乱弧菌 O1血清群20 。 CT是由五个CtxB组成的两部分毒素 亚单位围绕单个CtxA亚单位，并且负责与霍乱相关的快速电解质损失。 TCP是由tcp操纵子（ tcpABQCRDSTEF ）编码的IV型pilus ，并且涉及远端小肠的附着和定殖。 tcpA是tcp操纵子的第一个基因，其编码构建真菌8所必需的各个pilin亚单位。 ctxA的基因序列在经典和El Tor生物型之间是完全保守的，而ctxB和tcpA在两种生物型之间不同，但在每种生物型8中都是保守的。除了两个基本位置之外， ctxB在生物型之间是完全保守的（115和203）。在El Tor生物型中，胸腺嘧啶位于基因位置115和203，而经典生物型在这些碱基处含有胞嘧啶。 tcpA在每种生物型中都是完全保守的，但在生物型之间的多个基础上是不同的。这些遗传差异用作初级生物型鉴定标记，并且在测序包括这些位点的聚合酶链反应（PCR）扩增产物之后，可以将分离序列与野生型（WT）经典O395或WT E1 Tor N16961进行比较，以确定生物型背景的CT和TCP分别在给定的霍乱弧菌分离株中。 已经开发了许多方案来表征经典和El Tor生物型21,22,23之间的表型区别。多粘菌素B是一种肽抗生素，其在革兰氏阴性杆菌中损害外细胞膜的完整性通过多粘菌素B抗性测定可以观察到teria和多粘菌素B抗性21 。柠檬酸盐是克雷伯氏循环的主要底物，并且可以使用柠檬酸盐代谢测定法22来确定将柠檬酸盐代谢为唯一碳源的能力。 hapR编码霍乱弧菌 HapR中的全局调节子和主要群体感应调节因子，其结合各种启动子区域并调节基因和操纵子表达24 。 霍乱弧菌的一些致病菌株在hapR基因中具有天然存在的转录突变，导致毒力基因表达的密度依赖性调节丧失24,25 。使用牛奶琼脂培养基测量HapR调节的蛋白酶活性允许研究者鉴定特定分离物是否含有功能性HapR 23 。该溶血分析测试菌株分泌溶血红细胞的溶血酶的能力;溶血程度可以在血琼脂平板23上显现。运动性通常与霍乱弧菌中的毒力相关，可以使用运动性琼脂平板23进行分析。 Voges-Proskauer测定法测试菌株作为唯一碳源发酵葡萄糖并产生副产物乙偶姻21的能力 。随着El Tor变体的出现，很难预测任何给定的表型测定的结果，而没有广泛的基因型筛选，并且在推导V的生物型背景之前。 霍乱弧菌分离物，推荐进行这种测定23的装配，并将结果与表2中的参考菌株进行比较。 在这里，我们提出了一系列方案，集体利用上述方案基因型和表型检测用于更全面的表征V的方法 。 霍乱生物型此外，我们已经描述了已知V的基因型和表型区别。与常用的生物型参考菌株（WT经典O395，WT E1 Tor C6706和WT El Tor N16961; 表1 ）相比， 霍乱弧菌 El Tor变体（MQ1795和BAA-2163）。 El Tor变体的出现对先前使用的单一测定生物型表征方案的可靠性提出了挑战;然而，这种多重测定鉴定系统将允许更可靠地表征临床和环境V。 霍乱弧菌分离株

<table border="0" cellpadding="0" cellspacing="0" width="1403">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1 kb DNA Ladder</td>
			<td style="width:183px;">New England Biolabs</td>
			<td style="width:171px;">N3232S</td>
			<td style="width:683px;">https://www.neb.com/products/n3232-1-kb-dna-ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60% Glycerol</td>
			<td>Calbiochem</td>
			<td>356352</td>
			<td>http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade---CAS-56-81-5---Calbiochem,EMD_BIO-356352</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Becto, Dickinson and Co.</td>
			<td>214030</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar with brain-heart infusion</td>
			<td>Becto, Dickinson and Co.</td>
			<td>237500</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=237500&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Peqlab</td>
			<td>732-2789</td>
			<td>https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&#38;_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&#38;_dynSessConf=1766917479792147141&#38;targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&#38;lastLanguage=en&#38;/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&#38;_D%3AcurrentLanguage=+&#38;currentLanguage=en&#38;_D%3AlastLanguage=+&#38;_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anhydrous K2HPO4</td>
			<td>Fisher Scientific</td>
			<td>P288-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-phosphate-dibasic-anhydrous-crystalline-powder-certified-acs-fisher-chemical-5/p288500?searchHijack=true&#38;searchTerm=P288500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood Agar Plates</td>
			<td>Remel</td>
			<td>R01200</td>
			<td>Store at 4 &#176;C;&#160; http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>A73-500</td>
			<td>https://www.fishersci.com/shop/products/boric-acid-crystalline-certified-acs-fisher-chemical-6/a73500?searchHijack=true&#38;searchTerm=A73500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromothymol Blue</td>
			<td>Fisher Scientific</td>
			<td>B388-10</td>
			<td>https://www.fishersci.com/shop/products/bromothymol-blue-certified-acs-fisher-chemical/b38810?searchHijack=true&#38;searchTerm=B38810&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cirtric acid &#183;H2O</td>
			<td>Fisher Scientific</td>
			<td>S72836-3</td>
			<td>https://www.fishersci.com/shop/products/citric-acid-monohydrate-4/s728363#?keyword=s728363</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxynucleotide (dNTP) Solution Kit</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td>Store at -20 &#176;C;&#160; https://www.neb.com/products/n0446-deoxynucleotide-solutionset</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disodium EDTA</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>https://www.fishersci.com/shop/products/ethylenediaminetetraacetic-acid-disodium-salt-dihydrate-crystalline-certified-acs-fisher-chemical-7/s311500?searchHijack=true&#38;searchTerm=S311500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Clean &#38; Concentrator&#8482; -25 Kit</td>
			<td>Zymo Research</td>
			<td>D4007</td>
			<td>http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GelGreen Nucleic Acid Stain</td>
			<td>Biotium</td>
			<td>41005</td>
			<td>https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genesys 10SUV-VIS Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-208100</td>
			<td>https://www.thermofisher.com/order/catalog/product/840-208100?ICID=search-840-208100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentra Puregene Yeast/Bact. Kit</td>
			<td>Qiagen</td>
			<td>158567</td>
			<td>https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/gentra-puregene-yeastbact-kit/#orderinginformation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td>Corrosive;&#160; https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&#38;searchTerm=A144212&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-chloride-crystalline-certified-acs-fisher-chemical-4/p217500?searchHijack=true&#38;searchTerm=P217500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-phosphate-monobasic-crystalline-certified-acs-fisher-chemical-5/p285500?searchHijack=true&#38;searchTerm=P285500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td>Fisher Scientific</td>
			<td>P250-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&#38;searchTerm=P250500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Le Loop</td>
			<td>Decon Labs Inc.</td>
			<td>MP190-25</td>
			<td>http://deconlabs.com/products/leloop/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Le Stab</td>
			<td>Decon Labs Inc.</td>
			<td>MP186-5</td>
			<td>http://deconlabs.com/products/lestab/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?searchHijack=true&#38;searchTerm=M63500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Sub Cell GT Horizontal Electrophoresis System</td>
			<td>Bio Rad Labs</td>
			<td>1704406</td>
			<td>http://www.bio-rad.com/en-us/product/mini-sub-cell-gt-cell?WT.srch=1&#38;WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&#38;WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MR-VP Broth</td>
			<td>Difco</td>
			<td>216300</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>https://www.fishersci.com/shop/products/sodium-phosphate-dibasic-anhydrous-granular-powder-certified-acs-fisher-chemical-5/s374500?searchHijack=true&#38;searchTerm=S374500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-10</td>
			<td>https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&#38;searchTerm=S27110&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td>https://www.fishersci.com/shop/products/sodium-bicarbonate-powder-certified-acs-fisher-chemical-5/s233500?searchHijack=true&#38;searchTerm=S233500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaNH4HPO4&#183;4H2O</td>
			<td>Fisher Scientific</td>
			<td>S218-500</td>
			<td>https://www.fishersci.com/shop/products/sodium-ammonium-phosphate-tetrahydrate-crystalline-certified-fisher-chemical/s218500?searchHijack=true&#38;searchTerm=S218500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop Lite Spectrophtometer</td>
			<td>Thermo Scientific</td>
			<td>ND-LITE-PR</td>
			<td>https://www.thermofisher.com/order/catalog/product/ND-LITE-PR?ICID=search-ND-LITE-PR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonfat dry milk</td>
			<td>Nestle Carnation</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Becto, Dickinson and Co.</td>
			<td>211677</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (100 mm x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (150 mm x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875714</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&#38;searchTerm=FB0875714&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polymyxin B sulfate salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1004-10MU</td>
			<td>Store at 2-4 &#176;C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td>Store at -20 &#176;C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq Reaction Buffer</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td>Store at -20 &#176;C;&#160; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Cycler Bio-Rad C1000 Touch&#8482;&#160;</td>
			<td>Bio Rad Labs</td>
			<td>1840148</td>
			<td>http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&#38;_pageLabel=search_page&#38;sfMode=search&#38;sfStartNumber=1&#38;clearQR=true&#38;js=1&#38;searchString=1840148&#38;database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&#38;tabName=DIVISIONNAME</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triphenyltetrazolium chloride</td>
			<td>Alfa Aesar</td>
			<td>A10870</td>
			<td>https://www.alfa.com/en/catalog/A10870/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>https://www.fishersci.com/shop/products/tris-base-white-crystals-crystalline-powder-molecular-biology-fisher-bioreagents-7/bp1521?searchHijack=true&#38;searchTerm=BP1521&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8729;HCl</td>
			<td>Calbiochem</td>
			<td>9310</td>
			<td>http://www.emdmillipore.com/US/en/product/OmniPur-TRIS-Hydrochloride---CAS-1185-53-1---Calbiochem,EMD_BIO-9310-OP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>Becto, Dickinson and Co.</td>
			<td>211705</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becto, Dickinson and Co.</td>
			<td>212750</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-napthol</td>
			<td>MP Biomedicals</td>
			<td>204189</td>
			<td>http://www.mpbio.com/product.php?pid=05204189</td>
		</tr>
	</tbody>
</table>

laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates

水生革兰氏阴性菌霍乱弧菌是感染性胃肠道霍乱的病原体。由于这种疾病的全球流行和严重程度， V 。 霍乱弧菌已经在环境和实验室环境中得到广泛的研究，需要适当的维护和培养技术。古典和El Tor是组成V的两个主要生物型。 霍乱弧菌 O1血清群，各自显示独特的基因型和表型特征，为生物型表征提供可靠的机制，并需要不同的毒力诱导培养条件。不管任何给定的感染或爆发的致病菌株的生物型是什么，该疾病的标准治疗涉及补充有抗生素方案的补液治疗。然而，生物型分类对于实验室研究可能是必要的，并且可能在生物医学领域具有更广泛的影响。在2000年初，鉴定出了经典和El Tor生物型的基因型和表型性状的临床分离株。新鉴定的杂种，称为El Tor变体，使得临床和环境分离生物型鉴定变得比以前的传统单试验鉴定方案更复杂。除了描述V。 霍乱弧菌一般维护和培养技术，该手稿描述了一系列基于特定基因（ ctxB和tcpA ）基于PCR的遗传筛选和表型测定（多粘菌素B抗性，柠檬酸盐代谢，蛋白水解活性，溶血活性，运动性和葡萄糖代谢通过Voges- Proskauer测定）共同用于表征和/或区分经典和El Tor生物型。一起，这些测定提供了一种有效的系统方法，作为替代使用，或另外用于代表性的昂贵的劳动密集型实验V。 霍乱临床（和环境）分离株。

为了妥善维护和使用任何细菌菌株，建议知道感兴趣的菌株的倍增时间。这里，常用的V的变化的生长速率。通过生长曲线证实霍乱弧菌菌株，并使用线性回归计算近似倍数。 WT El Tor N16961和El Tor变体MQ1795比WT经典O395（〜2 h）显示更短的倍增时间（分别为〜1 h和〜1 h）（ 图1 ; 表2 ）。 <p class="jove_content" fo:keep-together.within-pa...

Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

该手稿除了进行一系列生物化学测定之外，还描述了适用于霍乱弧菌的维护技术，它们共同用于临床和环境V之间的快速可靠的分化。 霍乱弧菌生物型实验室设置。

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

laboratory-techniques-used-to-maintain-differentiate-biotypes-vibrio

Research

JoVE Journal

Immunology and Infection

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

10.8K 次观看.  Plymouth State University. 该手稿除了进行一系列生物化学测定之外，还描述了适用于霍乱弧菌的维护技术，它们共同用于临床和环境V之间的快速可靠的分化。 霍乱弧菌生物型实验室设置。 

视频: Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - A基因修改方法<em&gt;霍乱弧菌</em基于自然转化和FLP重组

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性<em&gt;隐</em&gt;和<em&gt;贾第虫属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

安全注意事项和操作步骤在（A）BSL-4实验室:1.生物安全4级实验室西服套房出入境手续

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...