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Article: Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format

TitleEvaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format
Authors
KeywordsInfluenza
Molecular diagnosis
Pandemic H1N1
Quantitative RT-PCR
Issue Date2010
PublisherElsevier BV. The Journal's web site is located at http://www.elsevier.com/locate/jviromet
Citation
Journal Of Virological Methods, 2010, v. 165 n. 2, p. 302-304 How to Cite?
AbstractThe recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E-01 to 1.68E-05 TCID50 per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E-05 TCID50 per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1. © 2010 Elsevier B.V.
Persistent Identifierhttp://hdl.handle.net/10722/125110
ISSN
2015 Impact Factor: 1.508
2015 SCImago Journal Rankings: 0.866
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
Area of Excellence Scheme of the University Grants Committee Hong KongAoE/M-12/06
National Institutes of Health (NIAID)HHSN266200700005C
Funding Information:

This work was supported by the Area of Excellence Scheme of the University Grants Committee Hong Kong Grant AoE/M-12/06 and the National Institutes of Health (NIAID contract HHSN266200700005C). We thank Brian Plew (Applied Biosystems) for technical support.

References
Grants

 

DC FieldValueLanguage
dc.contributor.authorCheung, TKWen_HK
dc.contributor.authorChin, AWHen_HK
dc.contributor.authorChan, KHen_HK
dc.contributor.authorSchumaker, Men_HK
dc.contributor.authorMak, PWYen_HK
dc.contributor.authorLeung, HSYen_HK
dc.contributor.authorWong, Aen_HK
dc.contributor.authorPeiris, JSMen_HK
dc.contributor.authorPetrauskene, OVen_HK
dc.contributor.authorPoon, LLMen_HK
dc.date.accessioned2010-10-31T11:11:56Z-
dc.date.available2010-10-31T11:11:56Z-
dc.date.issued2010en_HK
dc.identifier.citationJournal Of Virological Methods, 2010, v. 165 n. 2, p. 302-304en_HK
dc.identifier.issn0166-0934en_HK
dc.identifier.urihttp://hdl.handle.net/10722/125110-
dc.description.abstractThe recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E-01 to 1.68E-05 TCID50 per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E-05 TCID50 per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1. © 2010 Elsevier B.V.en_HK
dc.languageengen_HK
dc.publisherElsevier BV. The Journal's web site is located at http://www.elsevier.com/locate/jvirometen_HK
dc.relation.ispartofJournal of Virological Methodsen_HK
dc.subjectInfluenzaen_HK
dc.subjectMolecular diagnosisen_HK
dc.subjectPandemic H1N1en_HK
dc.subjectQuantitative RT-PCRen_HK
dc.titleEvaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized formaten_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0166-0934&volume=165&spage=302&epage=4&date=2010&atitle=Evaluation+of+novel+H1N1-specific+primer-probe+sets+using+commercial+RT-PCR+mixtures+and+a+premixed+reaction+stored+in+a+lyophilized+formaten_HK
dc.identifier.emailPeiris, JSM: malik@hkucc.hku.hken_HK
dc.identifier.emailPoon, LLM: llmpoon@hkucc.hku.hken_HK
dc.identifier.authorityPeiris, JSM=rp00410en_HK
dc.identifier.authorityPoon, LLM=rp00484en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1016/j.jviromet.2010.01.024en_HK
dc.identifier.pmid20138917-
dc.identifier.pmcidPMC2859313-
dc.identifier.scopuseid_2-s2.0-77951024323en_HK
dc.identifier.hkuros176294en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-77951024323&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume165en_HK
dc.identifier.issue2en_HK
dc.identifier.spage302en_HK
dc.identifier.epage304en_HK
dc.identifier.isiWOS:000277763700025-
dc.publisher.placeNetherlandsen_HK
dc.relation.projectControl of Pandemic and Inter-pandemic Influenza-
dc.identifier.scopusauthoridCheung, TKW=16229531100en_HK
dc.identifier.scopusauthoridChin, AWH=36243171200en_HK
dc.identifier.scopusauthoridChan, KH=7406034307en_HK
dc.identifier.scopusauthoridSchumaker, M=36244576200en_HK
dc.identifier.scopusauthoridMak, PWY=36022676500en_HK
dc.identifier.scopusauthoridLeung, HSY=36243721100en_HK
dc.identifier.scopusauthoridWong, A=35575315200en_HK
dc.identifier.scopusauthoridPeiris, JSM=7005486823en_HK
dc.identifier.scopusauthoridPetrauskene, OV=6602450688en_HK
dc.identifier.scopusauthoridPoon, LLM=7005441747en_HK
dc.identifier.citeulike6775459-

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