Dataset Information

Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.

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.

SUBMITTER: Hoehne M

PROVIDER: S-EPMC1524786 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.

Hoehne Marina M Schreier Eckart E

BMC infectious diseases 20060410

<h4>Background</h4>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 ...[more]

PMID: 16606447

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Project description:BackgroundBorrelia burgdorferi sensu lato (sl), the causative agent of Lyme borreliosis, is transmitted by ticks of the genus Ixodes as vector. For identification of Borrelia infections in ticks a TaqMan minor groove binder (MGB) probe-based quantitative real time PCR (qPCR) was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an Ixodes spp. positive control to verify successful DNA isolation. Besides qPCR, an ospA-specific conventional PCR for species-specific identification of B. spielmanii was established. Afterwards 1000 I. ricinus flagged in the city of Hanover, Germany, were investigated for B. burgdorferi sl infections followed by species identification. Furthermore, I. hexagonus ticks were investigated to proof applicability of the PCRs.ResultsQuantitative real time PCR (qPCR) identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-specific conventional PCR was also highly specific and showed no cross reactions with the other tested Borrelia species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was B. garinii, followed by B. afzelii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). 70.6% of I. ricinus were mono-infected, whereas 28.0% and 1.4% were infected with two and three Borrelia species, respectively. From 232 I. hexagonus collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with B. burgdorferi sl, which were identified as B. afzelii, B. garinii and B. spielmanii.ConclusionsThe evaluated qPCR to detect B. burgdorferi sl in Ixodes spp. is highly specific and sensitive. As a duplex qPCR including detection of Ixodes spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from the vector tick. Establishment of a B. spielmanii specific conventional PCR filled the gap in PCR identification of principal European Borrelia genospecies. Practical application showed that all European pathogenic Borrelia spp. were present in I. ricinus flagged in recreational areas of the city of Hanover and confirmed I. hexagonus as reservoir for pathogenic Borrelia spp.

Dataset Information

Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.

Publications

Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.

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