نشكر البرتو Severini وGoleski فانيسا للمساعدة في تطوير فحص والتعليقات الهامة على هذا المخطوط. وقد تم تمويل هذا العمل من قبل وكالة الصحة العامة في كندا لتقديم المساعدة وبرنامج البحوث الصناعية (المجلس القومي للبحوث في كندا). تم الحصول على دعم إضافي من جامعة ساسكاتشوان الصندوق النشر.

<ol>
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Gynecol. 108, 439-450 (2001).</li><li>Myer, L., Kuhn, L., Stein, Z. A., Wright, T. C., Denny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravaginal+practices,+bacterial+vaginosis,+and+women's+susceptibility+to+HIV+infection:+epidemiological+evidence+and+biological+mechanisms.">Intravaginal practices, bacterial vaginosis, and women's susceptibility to HIV infection: epidemiological evidence and biological mechanisms.</a> Lancet. Infect. Dis. 5, 786-794 (2005).</li><li>Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+recurrent+bacterial+vaginosis.">Managing recurrent bacterial vaginosis.</a> Sex. Transm. Infect. 80, 8-11 (2004).</li><li>Nugent, R. P., Krohn, M. A., Hillier, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+diagnosing+bacterial+vaginosis+is+improved+by+a+standardized+method+of+gram+stain+interpretation.">Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation.</a> J. Clin. Microbiol. 29, 297-301 (1991).</li><li>Ison, C. A., Hay, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+simplified+grading+of+Gram+stained+vaginal+smears+for+use+in+genitourinary+medicine+clinics.">Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics.</a> Sex. Transm. Infect. 78, 413-415 (2002).</li><li>Money, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+diagnosis+of+bacterial+vaginosis.">The laboratory diagnosis of bacterial vaginosis.</a> Can. J. Infect. Dis. Med. Microbiol. 16, 77-79 (2005).</li><li>Schellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing+of+the+chaperonin-60+universal+target+as+a+tool+for+determining+microbial+community+composition.">Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition.</a> Appl. Environ. Microbiol. 75, 2889-2898 (2009).</li><li>Spear, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+diversity+of+the+vaginal+microbiota+in+HIV-infected+and+HIV-uninfected+women+with+or+without+bacterial+vaginosis.">Comparison of the diversity of the vaginal microbiota in HIV-infected and HIV-uninfected women with or without bacterial vaginosis.</a> J. Infect. Dis. 198, 1131-1140 (2008).</li><li>Brotman, R. M., Ravel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ready+or+not:+the+molecular+diagnosis+of+bacterial+vaginosis.">Ready or not: the molecular diagnosis of bacterial vaginosis.</a> Clin. Infect. Dis. 47, 44-46 (2008).</li><li>Fredricks, D. N., Fiedler, T. L., Marrazzo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+bacteria+associated+with+bacterial+vaginosis.">Molecular identification of bacteria associated with bacterial vaginosis.</a> N. Engl. J. Med. 353, 1899-1911 (2005).</li><li>Dumonceaux, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+bacteria+associated+with+normal+microbiota+and+with+bacterial+vaginosis+in+vaginal+swabs+by+use+of+oligonucleotide-coupled+fluorescent+microspheres.">Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres.</a> J. Clin. Microbiol. 47, 4067-4077 (2009).</li><li>Molenkamp, R., van der Ham, A., Schinkel, J., Beld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+five+different+DNA+targets+by+real-time+Taqman+PCR+using+the+Roche+LightCycler480:+Application+in+viral+molecular+diagnostics.">Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics.</a> J. Virol. Meth. 141, 205-211 (2007).</li><li>Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., Rogers, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+phosphorothioate+primers+and+exonuclease+hydrolysis+for+the+preparation+of+single-stranded+PCR+products+and+their+detection+by+solid-phase+hybridization.">The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.</a> PCR. Methods. Appl. 3, 285-291 (1994).</li><li>Hill, J. E., Penny, S. L., Crowell, K. G., Goh, S. H., Hemmingsen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cpnDB:+A+Chaperonin+Sequence+Database.">cpnDB: A Chaperonin Sequence Database.</a> Genome. Res. 14, 1669-1675 (2004).</li><li>Bradshaw, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+of+Atopobium+vaginae+and+Gardnerella+vaginalis+with+bacterial+vaginosis+and+recurrence+after+oral+metronidazole+therapy.">The association of Atopobium vaginae and Gardnerella vaginalis with bacterial vaginosis and recurrence after oral metronidazole therapy.</a> J. Infect. Dis. 194, 828-836 (2006).</li><li>Dumonceaux, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+specific+bacterial+populations+in+complex+intestinal+communities+using+quantitative+PCR+based+on+the+chaperonin-60+target.">Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target.</a> J. Microbiol. Meth. 64, 46-62 (2006).</li></ol>

الإعلان عن أي تضارب في المصالح.

خصوصية توليد إشارة ذات أهمية حاسمة ، يجب أن تكون على ثقة من أن لاحظ إشارة تعكس حقا كشف amplicon المتولدة من ذلك الحي. يمكن لبرامج مثل PrimerPlex (الدوري Biosoft) على تصميم التحقيقات التي من شأنها أن يهجن بكفاءة ، لكنها قد تكون أو لا إلى الأنواع غير المستهدفة عبر هجن. عندما تستخدم با?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم قليل النوكليوتيد </td><td> شركة </td><td> تسلسل 1 </td></tr><tr><td> H279BP </td><td rowspan="3"> Invitrogen ، IDT ، أو غيرها من </td><td> البيوتين OEFO GAIIIIGCIGGIGAYGGIACIACIAC </td></tr><tr><td> H280 </td><td> YKIYKITCICCRAAICCIGGIGCYTT </td></tr><tr><td> H1612BP </td><td> البيوتين OEFO GAIIIIGCIGGYGACGGYACSACSAC </td></tr><tr><td> H1613 </td><td></td><td> CGRCGRTCRCCGAAGCCSGGIGCCTT </td></tr><tr><td colspan="3"> 1O ، phosphorothioate - C ، E ، G - phosphorothioate ؛ F ، phosphorothioate - A ، وأنا ، إينوزين ؛ Y ، C أو تي ، R ، A أو G ، K ، T أو G ، S ، C أو G. </td></tr></tbody></table> الجدول 1. متواليات من الجميع لتعديل [أليغنوكليوتيد] cpn60 PCR. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف</td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> 1 - 3 - الأثيل (3 - dimethylamiopropyl) carbodiimide حمض الهيدروكلوريك (EDC) </td><td> ثقب </td><td> 22980 </td><td></td></tr><tr><td> الخرز البوليسترين فلوري : MicroPlex المجهرية (Luminex) ، أو البيولوجية الصفيف COOH الخرزة (بيو راد) </td><td> Luminex أو بيو راد ، </td><td> بيو راد : 171 506xxx حيث xxx يناظر معرف حبة </td><td> حبات مغناطيسية أصبحت متاحة للاقتران قليل النوكليوتيد ويمكن أن تقدم مزايا معينة. لم يحاكموا من قبل هؤلاء الكتاب. </td></tr><tr><td> [أليغنوكليوتيد] القبض على (5 &#39;الأمينية تعديل C12) </td><td> Invitrogen ، IDT ، أو غيرها من </td><td> مختلف </td><td> المحلاة مستوى نقاء مقبولة. يتم توفير سلاسل من التحقيقات التقاط المستخدمة لتوصيف مسحات مهبلية في المخطوطة التي يستند إليها هذا البروتوكول 13. </td></tr><tr><td> T7نوكلياز خارجية </td><td> نيو انغلاند Biolabs </td><td> M0263S </td><td></td></tr><tr><td> Streptavidin - R - فيكوإيريترين (SA - PE) </td><td> Invitrogen </td><td> S - 866 </td><td> كن حذرا للحصول على درجة نقاوة عالية SA - PE ؛ ينصح هذا الرقم التسويقي </td></tr><tr><td> 96 Thermowell جيدا لوحات PCR </td><td> الصياد </td><td> CS006509 </td><td> تناسب كلا thermocycler 96 - جيدا وآلة BioPlex </td></tr><tr><td> Thermowell ختم حصيرة </td><td> الصياد </td><td> CS006555 </td><td> ويمكن إعادة استخدامها ؛ تغسل بالماء والصابون ، شطف جيدا ، والجافة </td></tr><tr><td> 5M TMAC </td><td> سيغما </td><td> T3411 </td><td></td></tr><tr><td> الحيوي الصفيف أو صك Luminex </td><td> بيو راد أو Luminex </td><td> بيو راد : 171-000201 </td><td></td></tr><tr><td> PrimerPlex برنامج لتصميم المسبار </td><td> رئيس الوزراء Biosoft </td><td> www.premierbiosoft.com </td><td> واقترح لتحقيق Luminex ديتوقيع ، على الرغم من يمكن استخدام منصات البرامج الأخرى </td></tr></tbody></table> الجدول 2. الكواشف معينة والمعدات. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> مكون </td><td> ميكروليتر / مقايسة </td><td> μl/100 المقايسات </td><td> التركيز النهائي </td></tr><tr><td> 10X PCR العازلة (Invitrogen) </td><td> 5 </td><td> 500 </td><td> 1X </td></tr><tr><td> 50 ملي MgCl 2 (Invitrogen) </td><td> 2.5 </td><td> 250 </td><td> 2.5 ملم </td></tr><tr><td> 10 ملي dNTP </td><td> 1 </td><td> 100 </td><td> 0.2 ملم كل </td></tr><tr><td> H279BP ، 25 ميكرومتر </td><td> 0.25 </td><td> 25 </td><td> 375 نانومتر </td></tr><tr><td> H1612BP ، 25 ميكرومتر </td><td> 0.75 </td><td> 75 </td><td> 125 نانومتر </td></tr><tr><td> H280 ، 25 ميكرومتر </td><td> 0.25 </td><td> 25 </td><td> 375 نانومتر </td></tr><tr><td> H1613 ،25 ميكرومتر </td><td> 0.75 </td><td> 75 </td><td> 125 نانومتر </td></tr><tr><td> ماء </td><td> 34 </td><td> 3400 </td><td> --- </td></tr><tr><td> المجاميع </td><td> 44.5 </td><td> 4450 </td><td></td></tr></tbody></table> مقترح الجدول 3. مخاليط لPCR مع UT تعديل الاشعال cpn60 (الجدول 1). تم تعيين مقايسة لمدة 5 ميكرولتر من الحمض النووي القالب و 0.5 ميكروليتر (2.5U) من الحمض النووي بوليميريز طق (Invitrogen). عادة ما يتم إعداد كميات كبيرة (على سبيل المثال يكفي لمدة 100 المقايسات) وتخزينها في -20 درجة مئوية.

multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres

التهاب المهبل الجرثومي (BV) هو متلازمة متعدد المكروبات المتكررة التي يتميز تغيير في مجهريات البقعة &quot;طبيعية&quot; من ​​يهيمن عليها الملبنة لمجهريات البقعة التي تهيمن عليها لعدد من أنواع البكتيريا ، بما في ذلك الغاردنريلة المهبلية ، Atopobium المهبل ، و 1-3 الآخرين. ويرتبط هذا الشرط مع مجموعة من نتائج سلبية على الصحة ، بما في ذلك اكتساب فيروس نقص المناعة البشرية 4 ، وأنه يمكن أن يكون من الصعب على إدارة سريريا 5. وعلاوة على ذلك ، اعتمد تشخيص BV على استخدام بقع غرام من المسحات المهبلية المسحة التي سجلت على معايير مختلفة 6،7 العددية. في حين أن هذا التشخيص غير بسيطة وغير مكلفة ، ويناسب المناطق ذات الموارد المحدودة ، ويمكن ان تعاني من مشاكل تتعلق التفسيرات الذاتية وأنها لا تعطي صورة تفصيلية للتكوين المهبل مجهريات البقعة 8. وقد كشفت مؤخرا جهود التسلسل عميقة ومتنوعة غنية مجهريات البقعة مع المهبليةواضح الخلافات بين عينات أخذت من الأفراد الذين يتم تشخيصهم مقارنة مع BV لأولئك الأفراد الذين يعتبرون 9،10 العادية ، مما أدى إلى تحديد عدد من الاهداف المحتملة للتشخيص الجزيئي لBV 11،12. وقد وفرت هذه الدراسات وجود ثروة من المعلومات المفيدة ، ولكن تسلسل عميق ليس بعد العملية كوسيلة تشخيصية في عملية إعداد سريرية. وقد وصفت لنا في الآونة الأخيرة وسيلة لتنميط بسرعة مجهريات البقعة المهبل في شكل متعدد باستخدام قليل النوكليوتيد ، إلى جانب الخرز الفلورسنت مع الكشف على منصة Luminex 13. هذا الأسلوب ، مثل الطرق الحالية وصمة عار على أساس غرام ، هو سريعة وبسيطة ولكنها تضيف ميزة إضافية لاستغلال المعرفة الجزيئية الناجمة عن التسلسل الدراسات في تصميم المسبار. هذا الأسلوب يوفر بالتالي وسيلة لملف كبير الكائنات الحية الدقيقة الموجودة في المسحة المهبلية التي يمكن أن تستخدم لتشخيص BV مع خصوصية عالية الحساسية وشركاتحمراء مكتوب لصبغة جرام بينما توفر معلومات إضافية حول وجود وفرة الأنواع وبطريقة شبه الكمي والسريع. هذا الأسلوب متعددة قابلة للتوسع إلى ما وراء نطاق المقايسات الحالية PCR الكمي للكائنات معينة ، والتي تقتصر حاليا على 5 أو 6 فحوصات مختلفة في عينة واحدة 14. الأهم من ذلك ، لا يقتصر على أسلوب للكشف عن البكتيريا في مسحات مهبلية ويمكن تكييفها بسهولة للملف بسرعة أي ما يقرب من المجتمع الميكروبي من الفائدة. على سبيل المثال ، بدأنا مؤخرا في تطبيق هذه المنهجية في تطوير أدوات تشخيصية للاستخدام في محطات معالجة مياه الصرف الصحي.

Watch this Scientific Journal Video about Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres at JoVE.com

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

نحن تصف طريقة متعددة للكشف عن الكائنات الدقيقة ضمن عينة باستخدام الخرز ، بالإضافة الفلورسنت قليل النوكليوتيد. غير المهجنة Amplicon من جميع الكائنات الحية داخل عينة على لجنة التحقيق ، إلى جانب الخرز. وتستخدم أداة أو Luminex بيو الصفيف إلى الاستعلام كل حبة حبة لنوع وإشارة التهجين.

كشف متعددة من البكتيريا الموجودة في مجمع العينات السريرية والبيئية باستخدام قليل النوكليوتيد ، بالإضافة نيون المجهرية

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

multiplex-detection-bacteria-complex-clinical-environmental-samples

Research

JoVE Journal

Immunology and Infection

16.1K Views.  Agriculture and Agri-Food Canada. نحن تصف طريقة متعددة للكشف عن الكائنات الدقيقة ضمن عينة باستخدام الخرز ، بالإضافة الفلورسنت قليل النوكليوتيد. غير المهجنة Amplicon من جميع الكائنات الحية داخل عينة على لجنة التحقيق ، إلى جانب الخرز. وتستخدم أداة أو Luminex بيو الصفيف إلى الاستعلام كل حبة حبة لنوع وإشار...

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

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.

باستخدام الفحص ميكروأري عموم الفيروسي (Virochip) إلى الشاشة العينات السريرية لمسببات الأمراض الفيروسية

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 ...

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

نيون استخدام الكيمياء المناعية للكشف عن الإدواردسيلة ictaluri في سمك السلور قناة ( أولا punctatus) عينات

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

تحديد نشاط أكلة العينات السريرية جسم

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

الكشف عن البكتيريا عن طريق DNAzymes Fluorogenic

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

التعرف السريع على البكتيريا سالبة الجرام من ثقافة الدم في مرق عن طريق MALDI-TOF الطيف الكتلي

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

في الكشف الموضعي للخلايا CD4 T Autoreactive في الدماغ والقلب عن طريق التوافق النسيجي الرئيسية الدرجة الثانية مجمع Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

الكشف الموضعي من البكتيريا داخل أنسجة البارافين جزءا لا يتجزأ من استخدام المسمى الديجوكسين DNA التحقيق استهداف 16S الريباسي

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

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.

استراتيجية السريع لعزل Faustoviruses الجديد من العينات البيئية عن طريق<em&gt; الدودي Vermamoeba</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.

تطوير اللعابية الأجسام المضادة المناعية المتعددة لقياس التعرض البشري للمسببات البيئية

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

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

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

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

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.