Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe
dc.contributor.author | Hoehne, Marina | |
dc.contributor.author | Schreier, Eckart | |
dc.date.accessioned | 2018-05-07T13:25:28Z | |
dc.date.available | 2018-05-07T13:25:28Z | |
dc.date.created | 2009-12-02 | |
dc.date.issued | 2006-04-10 | none |
dc.identifier.other | http://edoc.rki.de/oa/articles/retzRqdNBbiQ/PDF/26neP4juoVvFM.pdf | |
dc.identifier.uri | http://edoc.rki.de/176904/492 | |
dc.description.abstract | Background: 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.iso | eng | |
dc.publisher | Robert Koch-Institut, Infektionskrankheiten / Erreger | |
dc.subject | Animals | eng |
dc.subject | Humans | eng |
dc.subject | RNA | eng |
dc.subject | Viral/analysis | eng |
dc.subject | Sensitivity and Specificity | eng |
dc.subject | Caliciviridae Infections/diagnosis | eng |
dc.subject | DNA Primers | eng |
dc.subject | DNA Probes | eng |
dc.subject | Gastroenteritis/diagnosis | eng |
dc.subject | Norovirus/classification | eng |
dc.subject | Norovirus/genetics | eng |
dc.subject | Norovirus/isolation & purification | eng |
dc.subject | Reverse Transcriptase Polymerase Chain Reaction/methods | eng |
dc.subject.ddc | 610 Medizin | |
dc.title | Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe | |
dc.type | periodicalPart | |
dc.identifier.urn | urn:nbn:de:0257-1003077 | |
dc.identifier.doi | 10.1186/1471-2334-6-69 | |
dc.identifier.doi | http://dx.doi.org/10.25646/417 | |
local.edoc.container-title | BMC Infectious Diseases | |
local.edoc.fp-subtype | Artikel | |
local.edoc.type-name | Zeitschriftenartikel | |
local.edoc.container-type | periodical | |
local.edoc.container-type-name | Zeitschrift | |
local.edoc.container-url | http://www.biomedcentral.com/1471-2334/6/69 | |
local.edoc.container-publisher-name | BioMedCentral | |
local.edoc.container-volume | 6 | |
local.edoc.container-issue | 69 | |
local.edoc.container-year | 2006 |