Name: rapid molecular detection and differentiation of influenza viruses a and b
Uploaded: 2017-01-30T14:00:00
Description: Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).

ETIK ERKLÆRING: Brugen af ​​venstre-over kliniske prøver er godkendt og følger retningslinjerne fra Memorial Sloan Kettering Cancer Center Institutional Review Board. 1. Før Kørsel af Assay BEMÆRK: Influenza A og B-analysen er godkendt til nasopharyngeale podninger og nasopharyngeale podninger gemt i virustransportmediet. Svaberprøver er inkluderet i sættet og bør anvendes til optimal ydeevne. Men rayon, skum, strømmede, podninger eller polyester næseskrab kan også anvendes til at indsamle ...

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Influenzavirus er betydelige verdensomspændende årsager til sygelighed og dødelighed. Hurtig og nøjagtig diagnose af influenza er en af ​​de vigtigste nøgler til at styre influenza udbrud i løbet af respiratorisk sæson. Andre antigen-baserede tests er hurtige og lette at udføre; men de har lave følsomheder 13. På den anden side har de traditionelle molekylære test forbedret følsomhed, men kræver mere erfarne laboratorie teknologer at udføre og er dyrere. Influenza A og B-assay b...

Influenzavirusinfektioner resultere i en betydelig mængde af sygelighed og dødelighed hvert år 1, 2, 3. Ukompliceret influenza er præget af forfatningsmæssige og respiratoriske symptomer som feber, muskelsmerter, hovedpine, og ikke-produktiv hoste 4, 5. Ældre individer, unge børn, immunsvækkede patienter og patienter med underliggende co-morbiditet er på et højere risiko for alvorlige komplikationer som lungebetændelse, myocarditis, sygdom i centralnervesystemet, eller død 6, 7. Influenza-infektion er unik fra andre respiratoriske virus i at hurtig administration af antiviral behandling inden for 48 timer fra symptomdebut kan reducere sygdommens sværhedsgrad og længde 8. Hurtig identifikation af influenza har også væretvist sig at reducere brugen af unødvendige antibiotika 9, 10. Endvidere skal hospitalsindlagte patienter med influenza-infektioner placeres i isolerede rum med passende infektionskontrol forholdsregler. respiratoriske sygdomme forårsaget af ikke-influenzavirus, kan dog være svært at klinisk skelne fra influenza. Af denne grund, hurtig og nøjagtig diagnostisk test for influenza er meget vigtigt for den kliniske behandling patient. Flere analyser er tilgængelige til påvisning og identifikation af influenzavirus. Rapid influenza antigen test til påvisning (RIDTs) er meget udbredt i klinisk praksis som point-of-care test, fordi de er enkle at bruge og giver resultater inden for 15 til 30 minutter 11, 12; Men deres følsomheder varierer meget (10-80%), afhængigt af producenten, og befolkningen bliver testet, og den type influenza og subtype 13, 14, 15. Direkte fluorescens assays (DFAs) giver overlegne følsomhed end RIDTs, men behandlingstiden er større (~ 3 timer), og skal være afsluttet af dygtige teknologer 16, 17. Viral kultur har været den gyldne standard for diagnostik influenza og har forbedret følsomhed over begge RIDTs og DFAs 18. Dog kan influenza viral kultur tage alt fra 2-14 d til at fuldføre, faldende dens anvendelighed i medvirken patientbehandlingen 19. Endelig har nukleinsyreamplifikationsmetoder tests (NAAT) erstattet dyrkningsteknikker som den nye guldstandard i diagnostik influenza. NAAT anses for at have den største sensitivitet til detektering influenza i et par timer. Imidlertid NAAT er de dyreste assays og kræver specialiseret udstyr og teknikere til at udføre 5 <sop&gt;, 20, 21, 22, 23, 24, 25. Influenza A og B assayet beskrevet her er den første CLIA-frafaldes molekylær hurtige influenza test, der er let tilgængelige. Denne analyse fungerer ved at ansætte en nicking endonuklease Amplification Reaktion (NÆR), der bruger isoterm forstærkning med influenza-specifikke primere efterfulgt af måldetektering med molekylære beaconprober. Dette assay skelner influenza A fra B, kræver 2 min at oprette og behandle en prøve, og kræver i alt 15 min at fuldføre. Her præsenteres protokollen for Influenza A &amp; B-analysen. Derudover giver vi en prøve datasæt sammenligner ydeevnen af ​​influenza A og B assay på arkiveret nasopharyngeal vatpind (NPS) enheder, lagret i viral transportmedium (VTM) til en anden respiratorisk patogen panel assay.

<table border="0" cellpadding="0" cellspacing="0" width="660">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Alere i Instrument</td>
			<td style="width:64px;">Alere</td>
			<td style="width:303px;">NAT-000 (Global), NAT-024 (US)</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td style="width:303px;">425-000 (Global), 425-024 (US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere Universal Printer</td>
			<td>Alere</td>
			<td style="width:303px;">55115 (Global), alereiprinter&#160;(US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L precision pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L disposable pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viral transport medium</td>
			<td>Remel</td>
			<td>M4-RT</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid molecular detection and differentiation of influenza viruses a and b

Influenza er en smitsom luftvejssygdom forårsaget af influenzavirus A og B hos mennesker og forårsager en betydelig mængde af sygelighed og dødelighed hvert år. Influenza A og B assay var den første CLIA-frafaldes molekylær hurtige influenza test til rådighed. Den Influenza A og B-test virker ved at ansætte isoterm forstærkning med influenza-specifikke primere efterfulgt af måldetektering med molekylære beaconprober. Her, udførelsen af ​​influenza A og B assay på frosne, arkiverede nasopharyngeal podepind (NPS) prøver opbevaret i viral transportmedium (VTM) blev sammenlignet med en respiratorisk panel assay. Udførelsen af ​​Influenza A og B-assay blev evalueret ved sammenligning af resultaterne for luftvejene panel referencemetoden. Følsomheden for total influenzavirus A var 67,5% (95% CI (CI), 56,6-78,5), og specificiteten var 86,9% (CI, 71,0-100). For influenzavirus B-test, sensitivitet og specificitet var 90,2% (CI, 68,5-100) Og 98,8% (CI, 68,5-100), hhv. Dette system har den fordel, at en væsentlig kortere test tid end nogen anden i øjeblikket tilgængelige molekylære assay og den enkle, pipette-free procedure kører på en fuldt integreret, lukkede, små-fodaftryk system. Samlet, influenza A og B-assay evalueret i denne undersøgelse har potentiale til at tjene som en point-of-care hurtige influenza diagnostisk test.

I denne undersøgelse blev arkiveret NPS prøver indsamlet fra indlagte præsenterer med influenzalignende symptomer på Memorial Sloan-Kettering Cancer Center (MSKCC) under en influenza udbrud fra december 15, 2012 og 1. marts 2013. NPS prøver blev indgivet i 3 ml af VTM og testet som en del af rutinemæssig klinisk praksis med en molekylær analyse, som påviser et panel af respiratoriske vira (RP), herunder Influenza a, a-1, a-3, og B. i undersøgelsesperioden blev 3.675 NPS eksempla...

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Vi beskriver en hurtig, molekylær-baserede Influenza A og B-assay. Den Influenza-analysen registrerer hvert mål inden for 15 minutter ved at ansætte isoterm forstærkning med influenza-specifikke primere efterfulgt af måldetektering med molekylære beaconprober. Den Influenza A og B-analysen er brugervenlig og krævede minimale hands-on tid til at udføre.

Hurtig Molekylær Detection og Differentiering af Influenza Virus A og B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

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B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Fluorescensbaseret neuraminidase-inhiberingsassay i vurdering af den følsomhed over influenzavira til neuraminidaseinhibitoren Class of Antivirale

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Måling af Influenza neutraliserende antistof svar til A(H3N2) virus i menneskelige Sera af Microneutralization undersøgelser, som ved hjælp af MDCK-SIAT1 celler

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...