Name: multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres
Uploaded: 2011-10-23T14:00:00
Description: Watch this Scientific Journal Video about Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres at JoVE.com

हम परख विकास और इस पांडुलिपि पर महत्वपूर्ण टिप्पणी के साथ मदद के लिए अल्बर्टो Severini और वैनेसा Goleski धन्यवाद. इस काम कनाडा के जन स्वास्थ्य एजेंसी और औद्योगिक अनुसंधान सहायता कार्यक्रम (कनाडा के राष्ट्रीय अनुसंधान परिषद) द्वारा वित्त पोषित किया गया था. Saskatchewan प्रकाशन फंड के विश्वविद्यालय से अतिरिक्त सहायता प्राप्त हुई थी.

इस विधि Dumonceaux एट अल की रिपोर्ट में अनुसंधान के क्षेत्र में इस्तेमाल किया गया था. जे क्लीन. Microbiol 47, 4067-4077, doi: 10.1128/jcm.00112-09 (2009). समग्र प्रक्रिया चित्रण एक योजनाबद्ध आरेख चित्र 1 में प्रस्तुत किया है. <p...

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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>

संकेत पीढ़ी की विशिष्टता के महत्व का है, आप विश्वास होना चाहिए कि संकेत मनाया वास्तव में है कि जीव से उत्पन्न amplicon का पता लगाने के को दर्शाता है. PrimerPlex (प्रीमियर Biosoft) के रूप में सॉफ्टवेयर के लिए जांच है कि कुश?...

<table border="1"><tbody><tr><td> Oligonucleotide नाम </td><td> कंपनी </td><td> एक अनुक्रम </td></tr><tr><td> H279BP </td><td rowspan="3"> Invitrogen, IDT, या अन्य </td><td> बायोटिन - OEFO GAIIIIGCIGGIGAYGGIACIACIAC </td></tr><tr><td> H280 </td><td> YKIYKITCICCRAAICCIGGIGCYTT </td></tr><tr><td> H1612BP </td><td> बायोटिन - OEFO GAIIIIGCIGGYGACGGYACSACSAC </td></tr><tr><td> H1613 </td><td></td><td> CGRCGRTCRCCGAAGCCSGGIGCCTT </td></tr><tr><td colspan="3"> 1O, phosphorothioate सी, ई, phosphorothioate जी, एफ, phosphorothioate-A, मैं, inosine, वाई सी, या टी, आर, एक या जी, कश्मीर, टी, या जी, एस, सी या जी </td></tr></tbody></table> तालिका 1. सार्वभौमिक cpn60 पीसीआर के लिए संशोधित oligonucleotides के दृश्यों. <table border="1"><tbody><tr><td> अभिकर्मक के नाम</td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> 1-एथिल-3 (3 dimethylamiopropyl) carbodiimide एचसीएल (EDC) </td><td> पियर्स </td><td> 22980 </td><td></td></tr><tr><td> प्रतिदीप्त polystyrene मनकों: MicroPlex (Luminex) microspheres, या जैव Plex COOH मनका (जैव रेड) </td><td> Luminex या जैव - रेड </td><td> जैव रेड: 171 - 506xxx जहां xxx मनका पहचानकर्ता से मेल खाती है है </td><td> चुंबकीय मोती oligonucleotide युग्मन के लिए उपलब्ध होते जा रहे हैं और कुछ लाभ की पेशकश कर सकते हैं. क्या नहीं की कोशिश की गई इन लेखकों द्वारा. </td></tr><tr><td> कैद oligonucleotides (5 &#39;अमीनो C12 संशोधित) </td><td> Invitrogen, IDT, या अन्य </td><td> विभिन्न </td><td> Desalted शुद्धता स्तर स्वीकार्य है. कब्जा योनि swabs विशेषताएँ इस्तेमाल जांच के दृश्यों पांडुलिपि जिस पर इस प्रोटोकॉल 13 आधारित है में प्रदान की जाती हैं. </td></tr><tr><td> T7exonuclease </td><td> न्यू इंग्लैंड Biolabs </td><td> M0263S </td><td></td></tr><tr><td> Streptavidin-आर phycoerythrin (SA-पीई) </td><td> Invitrogen </td><td> S-866 </td><td> उच्च शुद्धता SA पीई प्राप्त सावधान रहो, इस सूची नंबर की सिफारिश की है </td></tr><tr><td> अच्छी तरह से 96 पीसीआर प्लेटें Thermowell </td><td> मछुआ </td><td> CS006509 </td><td> दोनों 96 अच्छी तरह thermocycler और BioPlex मशीन में फिट </td></tr><tr><td> Thermowell सील चटाई </td><td> मछुआ </td><td> CS006555 </td><td> किया जा सकता है फिर से इस्तेमाल किया, धोने, साबुन पानी के साथ अच्छी तरह कुल्ला और सूखी </td></tr><tr><td> 5M TMAC </td><td> सिग्मा </td><td> T3411 </td><td></td></tr><tr><td> जैव Plex या Luminex साधन </td><td> जैव रेड या Luminex </td><td> जैव रेड: 171-000201 </td><td></td></tr><tr><td> जांच डिजाइन के लिए PrimerPlex सॉफ्टवेयर </td><td> प्रीमियर Biosoft </td><td> www.premierbiosoft.com </td><td> Luminex जांच डे के लिए सुझावसाइन इन करते हैं, हालांकि अन्य सॉफ्टवेयर प्लेटफार्मों इस्तेमाल किया जा सकता है </td></tr></tbody></table> तालिका 2 विशिष्ट अभिकर्मकों और उपकरणों. <table border="1"><tbody><tr><td> घटक </td><td> / μl परख </td><td> μl/100 assays </td><td> अंतिम एकाग्रता </td></tr><tr><td> 10x पीसीआर बफर (Invitrogen) </td><td> 5 </td><td> 500 </td><td> 1x </td></tr><tr><td> 50 मिमी MgCl 2 (Invitrogen) </td><td> 2.5 </td><td> 250 </td><td> 2.5 मिमी </td></tr><tr><td> 10 मिमी dNTP </td><td> 1 </td><td> 100 </td><td> 0.2 मिमी प्रत्येक </td></tr><tr><td> H279BP, 25 सुक्ष्ममापी </td><td> 0.25 </td><td> 25 </td><td> 375 एनएम </td></tr><tr><td> H1612BP, 25 सुक्ष्ममापी </td><td> 0.75 </td><td> 75 </td><td> 125 एनएम </td></tr><tr><td> H280, 25 सुक्ष्ममापी </td><td> 0.25 </td><td> 25 </td><td> 375 एनएम </td></tr><tr><td> H1613,25 सुक्ष्ममापी </td><td> 0.75 </td><td> 75 </td><td> 125 एनएम </td></tr><tr><td> पानी </td><td> 34 </td><td> 3400 </td><td> --- </td></tr><tr><td> योग </td><td> 44.5 </td><td> 4450 </td><td></td></tr></tbody></table> तालिका 3 सुझाव पीसीआर के लिए संशोधित cpn60 केन्द्र शासित प्रदेशों प्राइमरों (तालिका 1) के साथ मिश्रण . परख टेम्पलेट डीएनए और Taq डीएनए पोलीमरेज़ (Invitrogen) के 0.5 μl (2.5U) के 5 μl के लिए सेट कर दिया जाता है. आम तौर पर बड़ी मात्रा में तैयार कर रहे हैं (जैसे 100 assays के लिए पर्याप्त) और -20 डिग्री सेल्सियस पर संग्रहीत

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.

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

हम oligonucleotide मिलकर फ्लोरोसेंट मनकों का उपयोग कर एक नमूना के भीतर सूक्ष्मजीवों का पता लगाने के के लिए एक मल्टीप्लेक्स विधि का वर्णन. एक नमूना के भीतर सभी जीवों से Amplicon जांच युग्मित मोती के एक पैनल को संकरित है. एक Luminex या जैव Plex साधन मनका प्रकार और संकरण संकेत के लिए प्रत्येक मनका क्वेरी के लिए प्रयोग किया जाता है.

मल्टीप्लेक्स परिसर नैदानिक ​​और पर्यावरण Oligonucleotide मिलकर प्रतिदीप्त microspheres का उपयोग कर नमूने में बैक्टीरिया की जांच

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

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

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

पर्यावरण के नमूनों से नई Faustoviruses के अलगाव का उपयोग के लिए एक तेजी रणनीति<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

एक लार एंटीबॉडी मल्टीप्लेक्स Immunoassay का विकास पर्यावरण रोगजनकों के लिए मानव एक्सपोजर को मापने के लिए

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

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

की जांच के लिए चुंबकीय उत्तेजक विधि<em&gt; त्रिचिनेल्ला</em&gt; स्नायु नमूने में लार्वा

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

मल्टीप्लेक्स परिसर नैदानिक ​​और पर्यावरण Oligonucleotide मिलकर प्रतिदीप्त microspheres का उपयोग कर नमूने में बैक्टीरिया की जांच

मल्टीप्लेक्स परिसर नैदानिक और पर्यावरण Oligonucleotide मिलकर प्रतिदीप्त microspheres का उपयोग कर नमूने में बैक्टीरिया की जांच