Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations
| Autor: | Breanna Caruso, Steven Jacobson, Irene Cortese, Giovanna S Brunetto, Kaylan Fenton, Joan Ohayon, Emily C. Leibovitch, Raya Massoud, Kory R. Johnson |
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| Rok vydání: | 2014 |
| Předmět: |
Adult
Male Proviral load Viral quantification T cell Clinical Neurology Biology Human T-lymphotropic virus Polymerase Chain Reaction Peripheral blood mononuclear cell Article Virus Cellular and Molecular Neuroscience Cerebrospinal fluid Virology Tropical spastic paraparesis medicine Humans Aged Digital droplet PCR Human T-lymphotropic virus 1 Reproducibility of Results virus diseases Middle Aged Viral Load biology.organism_classification medicine.disease HTLV-I Infections Paraparesis Tropical Spastic medicine.anatomical_structure Neurology DNA Viral Mutation Female Neurology (clinical) HAM/TSP Viral load |
| Zdroj: | Journal of Neurovirology |
| ISSN: | 1538-2443 1355-0284 |
| Popis: | An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL-real-time PCR-has multiple limitations, including increased inter-assay variability in compartments with low cell numbers, such as CSF. Therefore, in this study, we evaluated a novel technique for HTVL-1 PVL quantification, digital droplet PCR (ddPCR). In ddPCR, PCR samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, and queried for fluorescent signal. Due to the high number of independent events (droplets), Poisson algorithms are used to determine absolute copy numbers independently of a standard curve, which enables highly precise quantitation. This assay has low intra-assay variability allowing for reliable PVL measurement in PBMC and CSF compartments of both asymptomatic carriers (AC) and HAM/TSP patients. It is also useful for HTLV-1-related clinical applications, such as longitudinal monitoring of PVL and identification of viral mutations within the region targeted by the primers and probe. |
| Databáze: | OpenAIRE |
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