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

Vi takker Alberto Severini og Vanessa Goleski for hjælp med assay udvikling og kritiske kommentarer til dette manuskript. Dette arbejde blev finansieret af Public Health Agency of Canada og Industrial Research Assistance Program (National Research Council of Canada). Yderligere støtte blev indhentet fra University of Saskatchewan Offentliggørelse fonden.

Denne metode blev brugt i forskning, aflagde rapport i Dumonceaux et al. J. Clin. . Microbiol 47, 4067-4077, doi: 10.1128/jcm.00112-09 (2009). Et diagram viser den overordnede procedure er vist i Figur 1. 1. Bead kobling Her beskrives de metoder, der skal anvendes til kobling oligonukleotid sonder til polystyren Luminex perler (se tabel 2). Mængderne er tilpasset lidt for vurdering af nye fange sonder på forsøgsbasis, og dis...

<ol>
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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. 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Det særlige ved signalet generation er af afgørende betydning, og du skal være sikker på, at signalet observerede virkelig afspejler påvisning af amplikonet genereres fra den pågældende organisme. Software som PrimerPlex (Premier Biosoft) kan hjælpe med at designe sonder, der vil hybridisere effektivt, men de kan eller ikke kan på tværs af hybridisere til ikke-målarter. Når universelle PCR primere anvendes som beskrevet i denne protokol, er det vigtigt at huske på, at den genererede amplikonet repræsentere...

<table border="1"><tbody><tr><td> Oligonucleotid navn </td><td> Firma </td><td> Sequence 1 </td></tr><tr><td> H279BP </td><td rowspan="3"> Invitrogen, IDT, eller andre </td><td> Biotin-OEFO GAIIIIGCIGGIGAYGGIACIACIAC </td></tr><tr><td> H280 </td><td> YKIYKITCICCRAAICCIGGIGCYTT </td></tr><tr><td> H1612BP </td><td> Biotin-OEFO GAIIIIGCIGGYGACGGYACSACSAC </td></tr><tr><td> H1613 </td><td></td><td> CGRCGRTCRCCGAAGCCSGGIGCCTT </td></tr><tr><td colspan="3"> 1O, phosphorothioate-C, E, phosphorothioate-G, F, phosphorothioate-A, jeg, inosin, Y, C eller T, F, A eller G, K, T eller G, S, C eller G. </td></tr></tbody></table> Tabel 1. Sekvenser af den modificerede oligonukleotider for universel cpn60 PCR. <table border="1"><tbody><tr><td> Navn på reagenset</td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> 1-Ethyl-3-(3-dimethylamiopropyl) carbodiimide HCl (EDC) </td><td> Pierce </td><td> 22980 </td><td></td></tr><tr><td> Fluorescerende polystyren perler: MicroPlex Mikrokugler (Luminex) eller Bio-Plex COOH Bead (Bio-Rad) </td><td> Luminex eller Bio-Rad </td><td> Bio-Rad: 171-506xxx hvor xxx svarer til perlen identifikator </td><td> Magnetiske perler bliver tilgængelige for oligonucleotid kobling og kan give visse fordele. Er ikke blevet prøvet af disse forfattere. </td></tr><tr><td> Capture oligonukleotider (5 &#39;amino C12 modificeret) </td><td> Invitrogen, IDT, eller andre </td><td> forskellige </td><td> Desalted renhed niveau er acceptabelt. Sekvenser af indfangning sonder bruges til at karakterisere vaginal podninger findes i manuskriptet, som denne protokol er baseret på 13. </td></tr><tr><td> T7exonuclease </td><td> New England Biolabs </td><td> M0263S </td><td></td></tr><tr><td> Streptavidin-R-phycoerythrin (SA-PE) </td><td> Invitrogen </td><td> S-866 </td><td> Vær omhyggelig med at opnå høj renhed SA-PE, hvilket katalognummer anbefales </td></tr><tr><td> Lomme 96 brønde PCR-plader </td><td> Fisher </td><td> CS006509 </td><td> passer ind i både 96-godt thermocycler og Bioplex maskine </td></tr><tr><td> Lomme forsegling mat </td><td> Fisher </td><td> CS006555 </td><td> Kan genbruges, vaskes af med sæbevand, skylles godt, og tør </td></tr><tr><td> 5M TMAC </td><td> Sigma </td><td> T3411 </td><td></td></tr><tr><td> Bio-Plex eller Luminex instrument </td><td> Bio-Rad eller Luminex </td><td> Bio-Rad: 171-000201 </td><td></td></tr><tr><td> PrimerPlex software til sonde-design </td><td> Premier Biosoft </td><td> www.premierbiosoft.com </td><td> Foreslåede for Luminex sonde deunderskrive, selv om andre software platforme kan anvendes </td></tr></tbody></table> Tabel 2. Specifikke reagenser og udstyr. <table border="1"><tbody><tr><td> komponent </td><td> μl / assay </td><td> μl/100 assays </td><td> endelig koncentration </td></tr><tr><td> 10x PCR-buffer (Invitrogen) </td><td> 5 </td><td> 500 </td><td> 1x </td></tr><tr><td> 50 mM MgCl 2 (Invitrogen) </td><td> 2,5 </td><td> 250 </td><td> 2,5 mm </td></tr><tr><td> 10 mM dNTP </td><td> 1 </td><td> 100 </td><td> 0,2 mm hvert </td></tr><tr><td> H279BP, 25 μM </td><td> 0,25 </td><td> 25 </td><td> 375 nm </td></tr><tr><td> H1612BP, 25 μM </td><td> 0,75 </td><td> 75 </td><td> 125 Nm </td></tr><tr><td> H280, 25 μM </td><td> 0,25 </td><td> 25 </td><td> 375 nm </td></tr><tr><td> H1613,25 μM </td><td> 0,75 </td><td> 75 </td><td> 125 Nm </td></tr><tr><td> Vand </td><td> 34 </td><td> 3400 </td><td> --- </td></tr><tr><td> Totaler </td><td> 44,5 </td><td> 4450 </td><td></td></tr></tbody></table> Tabel 3. Foreslåede blandinger til PCR med modificeret cpn60 UT primere (tabel 1). Analysen er sat op til 5 mikroliter af template DNA og 0,5 μl (2.5U) af Taq DNA-polymerase (Invitrogen). Normalt store mængder er forberedt (f.eks tilstrækkeligt for 100 assays) og opbevares ved -20 ° C.

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

Bakteriel vaginose (BV) er en tilbagevendende polymikrobiale syndrom, der er karakteriseret ved en ændring i den &quot;normale&quot; mikrobiota fra Lactobacillus-domineret til en mikrobiota domineret af en række bakteriearter, herunder Gardnerella vaginalis, Atopobium vaginae, og andre 1-3. Denne betingelse er forbundet med en række negative sundhedsvirkninger, herunder hiv erhvervelse 4, og det kan være svært at håndtere klinisk 5. Endvidere har diagnosen BV har påberåbt sig brugen af Gram-farvning af vaginal podepind udstrygninger, der er scoret på forskellige numeriske kriterier 6,7. Mens denne diagnose er enkel, billig og velegnet til ressource-begrænsede indstillinger, kan det lider af problemer relateret til subjektive fortolkninger, og det giver ikke en detaljeret profil af sammensætningen af vaginal mikrobiota 8. Seneste dybe sekventering bestræbelser har afsløret en rig, varieret vaginal mikrobiota medklare forskelle mellem prøver taget fra enkeltpersoner, der er diagnosticeret med BV i forhold til de personer, der betragtes som normale 9,10, hvilket har resulteret i identifikation af en række potentielle mål for molekylær diagnostik af BV 11,12. Disse undersøgelser har givet et væld af nyttige oplysninger, men dybt sekvensering er endnu ikke praktisk som en diagnostisk metode i et klinisk miljø. Vi har for nylig beskrevet en metode til hurtigt at profilering af vaginal mikrobiota i en multiplex-format ved hjælp oligonucleotid-koblede lysstofrør perler med detektion på en Luminex platform 13. Denne metode, som den nuværende Gram-farvning-baserede metoder, er hurtig og enkel, men tilføjer yderligere fordel af at udnytte molekylær viden, der hidrører fra sekventering studier i sonde design. Denne metode giver derfor en måde at profilere store mikroorganismer, som er til stede i en vaginal podepind, der kan bruges til at diagnosticere BV med høj specificitet og følsomhed compared til Gram-farvning samtidig give yderligere oplysninger om arternes tilstedeværelse og overflod i en semi-kvantitativ og hurtig måde. Denne multipleks Metoden kan udvides langt ud over den vifte af aktuelle kvantitativ PCR assays for bestemte organismer, som for øjeblikket er begrænset til 5 eller 6 forskellige analyser i en enkelt prøve 14. Det er vigtigt, er metoden ikke begrænset til påvisning af bakterier i vaginal podninger og kan nemt tilpasses til hurtigt at profilen næsten enhver mikrobielle samfund af interesse. For eksempel har vi for nylig begyndt at anvende denne metode til udvikling af diagnostiske værktøjer til brug i renseanlæg.

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

Vi beskriver en multiplex metode til påvisning af mikroorganismer i en prøve ved hjælp oligonukleotid-koblede fluorescerende perler. Amplikon fra alle organismer i en prøve er hybridiseret til et panel af sonde-koblede perler. En Luminex eller Bio-Plex instrument bruges til at forespørge hver perle for perle type og hybridisering signal.

Multiplex Påvisning af Bakterier i komplekse kliniske og miljøprøver hjælp Oligonucleotid koblede Fluorescent Mikrokugler

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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