Zur Kurzanzeige

2006-04-10Zeitschriftenartikel DOI: 10.1186/1471-2334-6-69
Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe
dc.contributor.authorHoehne, Marina
dc.contributor.authorSchreier, Eckart
dc.date.accessioned2018-05-07T13:25:28Z
dc.date.available2018-05-07T13:25:28Z
dc.date.created2009-12-02
dc.date.issued2006-04-10none
dc.identifier.otherhttp://edoc.rki.de/oa/articles/retzRqdNBbiQ/PDF/26neP4juoVvFM.pdf
dc.identifier.urihttp://edoc.rki.de/176904/492
dc.description.abstractBackground: Due to an increasing number of norovirus infections in the last years rapid, specific, and sensitive diagnostic tools are needed. Reverse transcriptase-polymerase chain reactions (RT-PCR) have become the methods of choice. To minimize the working time and the risk of carryover contamination during the multi-step procedure of PCR the multiplex real-time RT-PCR for the simultaneous detection of genogroup I (GI) and II (GII) offers advantages for the handling of large amounts of clinical specimens. Methods: We have developed and evaluated a multiplex one-tube RT-PCR using a combination of optimized GI and GII specific primers located in the junction between ORF1 and ORF2 of the norovirus genome. For the detection of GI samples, a 3'-minor groove binder-DNA probe (GI-MGB-probe) were designed and used for the multiplex real-time PCR. Results: Comparable results to those of our in-house nested PCR and monoplex real-time-PCR were only obtained using the GI specific MGB-probe. The MGB-probe forms extremely stable duplexes with single-stranded DNA targets, which enabled us to design a shorter probe (length 15 nucleotides) hybridizing to a more conserved part of the GI sequences. 97% of 100 previously norovirus positive specimens (tested by nested PCR and/or monoplex real-time PCR) were detected by the multiplex real-time PCR. A broad dynamic range from 2 x 10(1) to 2 x 10(7) genomic equivalents per assay using plasmid DNA standards for GI and GII were obtained and viral loads between 2.5 x 10(2) and 2 x 10(12) copies per ml stool suspension were detected. Conclusion: The one-tube multiplex RT real-time PCR using a minor groove binder-DNA probe for GI is a fast, specific, sensitive and cost-effective tool for the detection of norovirus infections in both mass outbreaks and sporadic cases and may have also applications in food and environmental testing.eng
dc.language.isoeng
dc.publisherRobert Koch-Institut, Infektionskrankheiten / Erreger
dc.subjectAnimalseng
dc.subjectHumanseng
dc.subjectRNAeng
dc.subjectViral/analysiseng
dc.subjectSensitivity and Specificityeng
dc.subjectCaliciviridae Infections/diagnosiseng
dc.subjectDNA Primerseng
dc.subjectDNA Probeseng
dc.subjectGastroenteritis/diagnosiseng
dc.subjectNorovirus/classificationeng
dc.subjectNorovirus/geneticseng
dc.subjectNorovirus/isolation & purificationeng
dc.subjectReverse Transcriptase Polymerase Chain Reaction/methodseng
dc.subject.ddc610 Medizin
dc.titleDetection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe
dc.typeperiodicalPart
dc.identifier.urnurn:nbn:de:0257-1003077
dc.identifier.doi10.1186/1471-2334-6-69
dc.identifier.doihttp://dx.doi.org/10.25646/417
local.edoc.container-titleBMC Infectious Diseases
local.edoc.fp-subtypeArtikel
local.edoc.type-nameZeitschriftenartikel
local.edoc.container-typeperiodical
local.edoc.container-type-nameZeitschrift
local.edoc.container-urlhttp://www.biomedcentral.com/1471-2334/6/69
local.edoc.container-publisher-nameBioMedCentral
local.edoc.container-volume6
local.edoc.container-issue69
local.edoc.container-year2006

Zur Kurzanzeige