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Development of a highly sensitive real-time one step RT-PCR combined complementary locked primer technology and conjugated minor groove binder probe.

ABSTRACT: BACKGROUND: Enterovirus (EV) infections are commonly associated with encephalitis and meningitis. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patients, by ruling out other causes of disease. METHOD: To develop a sensitive and reliable assay for routine laboratory diagnosis, we developed a real-time one step reverse transcription polymerase chain reaction (RT-PCR) assay with minor groove binder probes and primers modified with complementary locked primer technology (TMC-PCR). We checked the sensitivity of the developed assay by comparing it to a previously published TaqMan probe real-time one-step RT-PCR (TTN-PCR) procedure using enteroviral isolates, Enterovirus Proficiency panels from Quality Control on Molecular Diagnostics (QCMD-2007), and clinical specimens from patients with suspected EV infections. RESULTS: One hundred clinical specimens from 158 suspected viral meningitis cases were determined to be positive by the TMC-PCR assay (63.29%), whereas only 60 were found to be positive by the TTN-PCR assay (37.97%). The positive and negative agreements between the TMC-PCR and TTN-PCR assays were 100% and 59.2%, respectively. CONCLUSION: This data suggest that the TMC-PCR assay may be suitable for routine diagnostic screening from patient suspected EV infection.

SUBMITTER: Hong J

PROVIDER: S-EPMC3142241 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Development of a highly sensitive real-time one step RT-PCR combined complementary locked primer technology and conjugated minor groove binder probe.

Hong JiYoung J Kang Byunghak B Kim Ahyoun A Hwang Seoyeon S Ahn Jinhee J Lee Sunhwa S Kim Jonghyen J Park Jae-Hak JH Cheon Doo-Sung DS

Virology journal 20110629

<h4>Background</h4>Enterovirus (EV) infections are commonly associated with encephalitis and meningitis. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patients, by ruling out other causes of disease.<h4>Method</h4>To develop a sensitive and reliable assay for routine laboratory diagnosis, we developed a real-time one step reverse transcription polymerase chain reaction (RT-PCR) assay with minor groove binder probes and primers modified with ...[more]

PMID: 21714898

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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

Development of a highly sensitive real-time one step RT-PCR combined complementary locked primer technology and conjugated minor groove binder probe.

Publications

Development of a highly sensitive real-time one step RT-PCR combined complementary locked primer technology and conjugated minor groove binder probe.

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