我们想确认为结核病的增长时间推移视频片段菲茨沃特肖恩和卡门Giannina月神科伦坡。马蒂罗珀我们感谢她彻底和极好的反馈，在编辑和共同创作的用户指南，从目前的协议大多是采取逐字。制作这个视频是由美国国立卫生研究院/ Fogarty国际中心的<a href="http://www.fic.nih.gov/">资助</a> http://www.fic.nih.gov/大卫AJ摩尔贡献威康信托在热带医学和读者临床研究员在英国伦敦帝国学院的传染病（奖学金获奖数量078067/Z/05）。马克F.布雷迪贡献作为美国国立卫生研究院/福格蒂国际中心研究员。

<ol><li> 准备库存解决方案<ol><li>磷酸盐缓冲库存<ol><li> 950毫升磷酸二氢钾解决方案（磷酸钾溶于千毫升蒸馏水二氢9.07克），搅拌混合950毫升钠磷酸氢二钠溶液（溶于千毫升蒸馏水磷酸钠二元9.47克）;保持每一个解决方案，回到50毫升调整如有必要的pH值</li><li>调节pH值至6.8 ± 0.2：添加钠磷酸氢二钠溶液，以提高pH值;添加磷酸二氢钾溶液，以较低的pH值</li><li>高压灭菌器在121-124 ° C下15分钟消毒</li><li>铠甲一个100μL分装在培养皿中，并培育营养琼脂培养基上的磷酸盐缓冲溶液，在37 ° C，48小时，以确认无菌</li><li>存放在冰箱中2-8℃长达一个月 注 ：每个痰标本需要磷酸盐缓冲液10毫升 </li></ol></li><li>去污股票<ol><li>合并相同体积的氢氧化钠溶液（氢氧化钠溶解在200ml无菌蒸馏水8.0克）和柠檬酸钠溶液（柠檬酸钠在200ml无菌蒸馏水蒸馏水5.8克） </li><li> 15分钟组合在121-124 ° C的反应釜</li><li>存放在冰箱中2-8℃长达一个月 注 ：每个痰标本需要净化股票2毫升 </li></ol></li><li>文化传媒股票<ol><li>添加到无菌蒸馏水900毫升casitone和5.9克7H9培养基粉的甘油和1.25克3.1毫升;不断搅拌混合，直到完全溶解</li><li> 15分钟在121-124 ° C的高压灭菌器</li><li>冷却和抗生素的解决方案和内部控制划分为准备进入4.5毫升等分在无菌培养基无菌16X 100毫米的样品制备的玻璃试管，10.8毫升等分</li><li>在37 ° C孵育48小时，以验证不育（浊度缺乏） </li><li>在2-8 °与电容C商店紧闭长达1个月 注 ：每个痰标本需要一个管7H9培养基含有4.5毫升 </li></ol></li><li>抗生素的库存解决方案<ol><li> A.异烟肼股票（8 mg / ml的） <ol><li>溶解完全在2.5毫升无菌蒸馏水20毫克异烟肼</li><li> 0.2μm的注射器过滤器过滤水溶剂</li><li>在20μL无菌微型离心管分装于-20 ° C存储长达6个月 注 ：每个存储20μL等分是足够100个样本（包括浪费） </li></ol></li><li> B.利福平的股票（8 mg / ml的） </li></ol><ol><li>完全溶解20毫克利福平1.25毫升二甲基亚砜</li><li>添加1.25毫升无菌蒸馏水和混合</li><li>有机溶剂过滤0.2μm的注射器过滤器</li><li>在20μL无菌离心管分装于-20 ° C存储长达6个月 注 ：每个存储20μL等分是足够100个样本（包括浪费） </li></ol></li></ol></li><li> 准备工作的解决方案 <ol><li>除污工作解决方案<ol><li> NALC晶体溶解在每一个净化所需的解决方案20毫升0.1克 注 ：每个样本需要2毫升去污液 注 ：放弃任何氢氧化钠NALC去污解决方案，仍然未使用24小时后，作为NALC随着时间的推移失去mucolytic活动 </li></ol></li><li>文化媒体工作的解决方案<ol><li>列明： <ol><li> 1管与7H9培养基每痰标本进行处理，再加上每板1管（阴性对照列） </li><li> 2 7H9培养基为阳性对照管（3如果monoresistant株） </li><li> 1-2与抗生素溶液的制备10.8毫升7H9试管</li></ol></li><li>每个样品管中添加0.5毫升OADC 7H94.5毫升</li><li>添加1.2毫升OADC管10.8毫升7H9 </li><li>抛开5毫升7H9 - OADC为阳性对照，与管12毫升7H9 - OADC要使用抗生素溶液的配制（不需要潘塔）（S）2管</li><li>重组潘塔，并添加0.1毫升到每个样品管和阴性对照管（7H9 - OADC潘塔：总体积=5.1毫升） 注 ：潘塔（7H9 - OADC潘塔 ）的完全培养基用于痰标本和阴性对照;没有潘塔使用7H9 - OADC为阳性对照，抗生素溶液的制备 </li></ol></li><li>抗生素工作方案<ol><li> 1。参见图1，在未使用的24孔板抗生素的工作方案的步骤 注 ： 准确的药敏结果，最后的抗生素浓度是至关重要的 。 下面的过程是为laboratories配备与微量。如果不可用微量，结核菌素注射器可以使用不同的稀释系列指南附录3中所述的用户在modsperu.org 注意 ： 请不要再冻结或重新使用抗生素的工作或股票解决方案，作为药物活性 ，可能会丢失，丢弃所有未使用的抗生素解决方案，在处理天图1月底。使抗生素工作方案（用户指南在modsperu.org） </li></ol></li></ol></li><li> 痰标本和板的制备 <ol><li>去污<ol><li>痰标本的放置到一个15ml离心管（如果2毫升少，弥补至2ml，用磷酸盐缓冲液，如果有更多的，只能使用2毫升） </li><li>加2ml氢氧化钠NALC解决方案</li><li>第管紧紧地和20秒的旋涡;反转管，以确保氢氧化钠NALC解决方案接触整个管内表面和盖子</li><li>静置至少15分钟 - 如果样品特别是粘稠的，可以延长几分钟。为了避免过度治疗， 不应超过20分钟 </li><li>填写用磷酸盐缓冲液（pH值6.8）管14毫升以消除碱和终止的净化过程，拌匀反相管的4倍</li><li>离心15分钟（在modsperu.org用户指南附录2方程要达到3000克每分钟旋转3000克） </li><li>小心倒出上清液进入一个液体废物的容器，用10％的次氯酸钠或其他合适的消毒剂，并保留沉淀</li></ol></li><li>最终样品悬浮液的制备和备份<ol><li>使用7H9 - OADC潘塔（从试管中5.1毫升），样品颗粒悬浮在一个总量各2ml离心管中的巴斯德吸管;拌匀</li><li>在2-8℃的样品悬挂系统和存储在离心管中取出作为备份1毫升</li><li>添加第二个1ml到样品悬挂其余7H9 - OADC潘塔管;拌匀。这是最后的样品溶液，电镀</li></ol></li><li>放入24孔板的样品悬浮液<ol><li>将900μl的最终样品悬挂到每个列的4井在24孔板</li><li>额外的样品重复，直到第3列，该板块的所有列，除填补（或直到所有样本均镀） </li><li> 7H9 - OADC介质的地方900μl样本没有在每个样品盘的第3列的4口井（消极的内部控制） </li></ol></li><li>样品中添加抗生素<ol><li>使用多道移液器，认真填写7H9 - OADC和抗生素的工作解决方案（2列抗生素稀释板见图1）井100μL4要诀</li><li> 不接触板或样品添加100μL等分列1井900μl样品溶液</li><li>重复，直到所有的列已经收到了额外的介质的100μL（无毒品井）或抗生素的解决方案（包括阴性对照列3） </li><li>关闭盖放置在一个密封的聚乙烯袋（自封袋）和密封板（ 袋是不是从这时开始再次打开 ） </li><li>孵育温度37 ° C（CO 2的浓缩，是没有必要） </li></ol></li></ol></li><li> 质量控制 <ol><li> 阴性对照，每盘上运行。阴性对照列的任何水井中的任何增长的存在表明交叉污染，因此整个板块必须被丢弃，重新镀金的样品。 </li><li> 阳性对照是运行在一个单独的板块，在这一天结束 。一个完全对药物敏感的菌株是在一列和应变耐INH和RIF是运行在另一列运行。如果有运行的多药耐药株的关注，既是异烟肼monoresistant应变和一个RIF的monoresistant应变可以在运行的敏感和多药耐药株。所有阳性对照应在无毒井的增长表明，媒体支持经济增长。为了证明是正确的药物浓度，不应成长敏感品系在INH或RIF井，但耐多药菌株增长也增长在INH和RIF井。 </li></ol></li><li> 读板 <ol><li>随着电镀一天“0天”，读板从5日开始，并如果是负数，第5天的阅读是每一天，直到第14天，然后从第15天，每2-3天（或根据工作量的替代天） -21。板块研究上与10倍的目标倒光镜</li><li> 培养阳性 ，其中有两个或两个以上的菌落形成单位（&gt; 2 CFU）无毒井。早期的结核分枝杆菌生长，看起来像小弯逗号或螺旋（5-9天）。集落形成通常的进展，以“线”或“缠结”（上modsperu.org看到的图片库） </li><li>当天的文化是积极的无毒2井，含药井应阅读。 任何一种药物的存在增长表明该药物的抵抗力。 注 ：如果含药井阅读&gt; 1周后做一个积极的文化是在无毒井指出，这未必代表突破增长阻力是偶尔看到 </li><li>样品21天不积极被认为是负面的文化</li><li>浑浊的水井是一个污染繁茂的结果，而不会导致结核分枝杆菌生长在混浊</li><li>在完成21天的文化可能会被丢弃。文化留在各自的密封袋，被放置在高压灭菌袋在121-124 ° C灭菌45-60分钟</li></ol></li></ol>

<ol>
	<li>Arias, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+microscopic-observation+drug-susceptibility+assay+for+detection+of+tuberculosis.">Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis.</a> Clin Infect Dis. 44, 674-674 (2007).</li><li>Caviedes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+efficient+detection+and+drug+susceptibility+testing+of+Mycobacterium+tuberculosis+in+sputum+by+microscopic+observation+of+broth+cultures.+The+Tuberculosis+Working+Group+in+Peru.">Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru.</a> J Clin Microbiol. 38, 1203-1203 (2000).</li><li>Caviedes, L., Moore, 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=Introducing+MODS:+a+low-cost,+low-tech+tool+for+high-performance+detection+of+tuberculosis+and+multidrug+resistant+tuberculosis.">Introducing MODS: a low-cost, low-tech tool for high-performance detection of tuberculosis and multidrug resistant tuberculosis.</a> Indian J Med Microbiol. 25, 87-87 (2007).</li><li>Caws, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+MODS+culture+technique+for+the+diagnosis+of+tuberculous+meningitis.">Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis.</a> PLoS ONE. 2, e1173-e1173 (2007).</li><li>Ejigu, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic-observation+drug+susceptibility+assay+provides+rapid+and+reliable+identification+of+MDR-TB.">Microscopic-observation drug susceptibility assay provides rapid and reliable identification of MDR-TB.</a> Int J Tuberc Lung Dis. 12, 332-332 (2008).</li><li>Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+occupational+tuberculosis+in+National+Tuberculosis+Programme+laboratories+in+Korea.">Risk of occupational tuberculosis in National Tuberculosis Programme laboratories in Korea.</a> Int J Tuberc Lung Dis. 11, 138-138 (2007).</li><li>Mello, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+microscopic+observation+drug+susceptibility+assay+for+detection+of+Mycobacterium+tuberculosis+resistance+to+isoniazid+or+rifampin.">Clinical evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to isoniazid or rifampin.</a> J Clin Microbiol. 45, 3387-3387 (2007).</li><li>Moore, 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=Future+prospects+for+the+MODS+assay+in+multidrug-resistant+tuberculosis+diagnosis.">Future prospects for the MODS assay in multidrug-resistant tuberculosis diagnosis.</a> Future Microbiol. 2, 97-97 (2007).</li><li>Moore, 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=Infrequent+MODS+TB+culture+cross-contamination+in+a+high-burden+resource-poor+setting.">Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting.</a> Diagn Microbiol Infect Dis. 56, 35-35 (2006).</li><li>Moore, 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=Microscopic-observation+drug-susceptibility+assay+for+the+diagnosis+of+TB.">Microscopic-observation drug-susceptibility assay for the diagnosis of TB.</a> N Engl J Med. 355, 1539-1539 (2006).</li><li>Moore, 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=Microscopic+observation+drug+susceptibility+assay,+a+rapid,+reliable+diagnostic+test+for+multidrug-resistant+tuberculosis+suitable+for+use+in+resource-poor+settings.">Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings.</a> J Clin Microbiol. 42, 4432-4432 (2004).</li><li>Moore, D. A., Roper, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+smear-negative+tuberculosis+in+people+with+HIV/AIDS.">Diagnosis of smear-negative tuberculosis in people with HIV/AIDS.</a> Lancet. 370, 1033-1033 (2007).</li><li>Oberhelman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+recovery+of+Mycobacterium+tuberculosis+from+children+using+the+microscopic+observation+drug+susceptibility+method.">Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method.</a> Pediatrics. 118, e100-e100 (2006).</li><li>Palomino, J. C., Martin, A., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MODS+assay+for+the+diagnosis+of+TB.">MODS assay for the diagnosis of TB.</a> N Engl J Med. 356, 188-189 (2007).</li><li>Park, W. G., Bishai, W. R., Chaisson, R. E., Dorman, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+microscopic+observation+drug+susceptibility+assay+in+drug+susceptibility+testing+for+Mycobacterium+tuberculosis.">Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis.</a> J Clin Microbiol. 40, 4750-4750 (2002).</li><li>Shiferaw, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microscopic+observation+drug+susceptibility+assay+for+detection+of+multidrug-resistant+Mycobacterium+tuberculosis.">Evaluation of microscopic observation drug susceptibility assay for detection of multidrug-resistant Mycobacterium tuberculosis.</a> J Clin Microbiol. 45, 1093-1093 (2007).</li><li>Tovar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+diagnosis+of+pleural+tuberculosis+using+the+microscopic-+observation+drug-susceptibility+technique.">Improved diagnosis of pleural tuberculosis using the microscopic- observation drug-susceptibility technique.</a> Clin Infect Dis. 46, 909-909 (2008).</li><li>Vargas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+sputum-scarce+HIV-associated+pulmonary+tuberculosis+in+Lima,+Peru.">Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru.</a> Lancet. 365, 150-150 (2005).</li></ol>

MODS的实验是针对资源有限的环境。这是第一次，MODS的带来的快速液体培养法检测肺结核的能力和资源有限的环境测试3元多药耐药结核病。 MODS是一个非专有的，迭代的方法，和MODS的社会总是在其他实验室已设法使改善。 是一个经常性的关注，是因为液体可以溢出或雾化的液体介质中的结核病文化的生物安全。我们相信，MODS的检测是比任何实验，涉及的间接药敏试验BIOSAFE间接药敏试验，因为涉及操纵分?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Refrigerator/ freezer</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to store pre-prepared broth and antibiotic stocks</td>
</tr>
<tr>
<td>Vortex</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to aid sputum decontamination</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for sputum concentration; capable of reaching 3000 g; does not need to be refrigerated, but MUST be biosafe (buckets can be sealed)</td>
</tr>
<tr>
<td>Incubator (37 degree C)</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for culture; need not be CO2 enriched</td>
</tr>
<tr>
<td>Inverted light microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to read MODS plates</td>
</tr>
<tr>
<td>Autoclave</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to sterilize media, PBS and used plates</td>
</tr>
<tr>
<td>Balance</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to weigh isoniazid, rifampicin and NALC</td>
</tr>
<tr>
<td>Middlebrook 7H9 broth (Difco)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0713-17-9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Casitone (pancreatic digest casein)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0259&#x2010;17&#x2010;9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Glycerol (glycerin) lyophilized</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G&#x2010;33&#x2010;500</td>
<td>500ml/bottle; culture media base</td>
</tr>
<tr>
<td>PANTA (Antibiotic mixture lyophilized BD) </td>
<td>Reagent</td>
<td>Fisher</td>
<td>B4345114</td>
<td>6 bottles/pack; antibiotic media supplement</td>
</tr>
<tr>
<td>OADC (Middlebrook OADC enrichment BD)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>B11886</td>
<td>10 x 20ml/pack; nutritional media supplement</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (Hibri-Max)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D-2650</td>
<td>100ml/bottle; to prepare rifampicin stock</td>
</tr>
<tr>
<td>Antibiotic stocks: isoniazid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I-3377</td>
<td>50gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Antibiotic stocks: rifampicin</td>
<td>Reagent</td>
<td>Sigma 99</td>
<td>R-3501</td>
<td>1gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Sodium hydroxide (pellets)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>221465</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium citrate (trisodium salt dihydrate)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S-4641</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>N-acetyl-L-cysteine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7250</td>
<td>50gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Potassium Phosphate Monobasic crystal. KH2PO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0662</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium Phosphate Dibasic, anhydrous. Na2HPO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0876</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium hypochlorite</td>
<td>Reagent</td>
<td>household bleach</td>
<td>&nbsp;</td>
<td>to discard contaminated waste</td>
</tr>
<tr>
<td>15ml centrifuge tubes (polypropylene 15ml Falcon 35&#x2010;2096)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>14&#x2010;959&#x2010;49B</td>
<td>500ea/case; for sputum decontamination and concentration</td>
</tr>
<tr>
<td>24 well plates (Plates Tissue 24 wells BD Falcon 35&#x2010;3047)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>50 plates/case; for culture and reading</td>
</tr>
<tr>
<td>Sealable polythene bags 6 X 6 &#x201C; (ziplock)</td>
<td>Consumable</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for biosecurity to contain 24 well plate</td>
</tr>
<tr>
<td>Glass tubes with lid (16 x 100mm and 18 x 145mm)</td>
<td>Consumable</td>
<td>VWR</td>
<td>47729-583</td>
<td>500 tubes/case; to store aliquots of prepared broth</td>
</tr>
<tr>
<td>Screw cap microcentrifuge tubes (1.5ml)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>05&#x2010;669&#x2010;22</td>
<td>1000ea/case; to store aliquots of antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (aqueous solvents) Syringe filter Millex blue</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGL 025 OS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (organic solvents) Syringe filter Millex yellow</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGV 033 RS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>Disposable Pasteur pipettes borosilicate glass 9&#x2033;</td>
<td>Consumable</td>
<td>Fisher</td>
<td>13&#x2010;678&#x2010;20C</td>
<td>720ea/case; to mix PANTA with media mix</td>
</tr>
<tr>
<td>Aerosol barrier tips 1000&#x2010;1300&#x03BC;l</td>
<td>Consumable</td>
<td>Fisher</td>
<td>02&#x2010;707&#x2010;51</td>
<td>1000ea/pk; to dispense media into plate</td>
</tr>
<tr>
<td>USA Scientific Tips One 1&#x2010;200&#x03BC;l yellow tips</td>
<td>Consumable</td>
<td>Fisher</td>
<td>1111&#x2010;0006</td>
<td>1000 tips/bag; to dilute antibiotic stocks</td>
</tr>		</tbody>
		</table>
		</div>

the mods method for diagnosis of tuberculosis and multidrug resistant tuberculosis

活动性肺结核患者（TB）的感染每年10-15其他人，使诊断活动性结核病，既治愈病人，并防止新的感染至关重要。此外，耐药结核病（MDRTB）的出现意味着停止耐药菌株的传播耐药性的检测是必要的。显微镜观察药物敏感性（MODS）的检测方法是一种低成本，低技术含量的高性能的检测结核病和MDRTB工具。结核分枝杆菌（MTB）的增长速度比2固体培养基上，在液体介质）微观山地车的增长可以被检测到，早在液体介质等待宏观固体培养基上菌落外观，并MODS的检测是基于三个原则：1）增长是MTB的特点，使得它从非典型分枝杆菌或真菌或细菌污染3）杰出的药物异烟肼和利福平可分为MODS的检测纳入到允许MDRTB同时直接检测，这样就不必为亚文化执行间接的药敏试验。痰涂片的灵敏度低，长期拖延，直到诊断与固体培养基培养，与现有的液体培养基中培养的方法，成本过高，而且需要做间接药敏试验检测MDRTB亚文化阻碍了竞争的目前诊断。的非专有MODS的方法相反，一个结核病和MDRTB高灵敏度，是一个较快的文化方法，同时药物易感性测试为MDRTB，只是根据美元的结核病测试3资源有限的环境和MDRTB。

Watch this Scientific Journal Video about The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis at JoVE.com

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

显微镜观察药物敏感性（MODS）的检测方法是一种低成本，低技术含量的高性能的检测结核病（TB）和耐多药结核病（MDRTB）工具。本视频介绍MODS的液体培养基中培养方法。

MODS的方法和诊断结核病耐药结核病

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

the-mods-method-for-diagnosis-tuberculosis-multidrug-resistant

Research

JoVE Journal

Biology

18.9K 次观看.  The Warren Alpert Medical School of Brown University. 显微镜观察药物敏感性（MODS）的检测方法是一种低成本，低技术含量的高性能的检测结核病（TB）和耐多药结核病（MDRTB）工具。本视频介绍MODS的液体培养基中培养方法。

视频: The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

注射的。 stephensi胚胎生成抗药性疟疾的蚊子

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

科研

生物学

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...