Dataset Information

Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.

ABSTRACT: Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies.

SUBMITTER: Trombley AR

PROVIDER: S-EPMC2861391 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.

Trombley Adrienne R AR Wachter Leslie L Garrison Jeffrey J Buckley-Beason Valerie A VA Jahrling Jordan J Hensley Lisa E LE Schoepp Randal J RJ Norwood David A DA Goba Augustine A Fair Joseph N JN Kulesh David A DA

The American journal of tropical medicine and hygiene 20100501 5

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the ...[more]

PMID: 20439981

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Project description:BackgroundViral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection.ResultsPrimers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens.ConclusionsOverall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs.

Project description:BackgroundAccurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories.MethodsHere we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays.ResultsThis ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10-5 (i.e. one parasite copy in 105 host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence (R2?=?0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians.ConclusionsWe developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories.

Dataset Information

Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.

Publications

Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.

Similar Datasets

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