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.
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.
Viral Immunology Section, Neuroimmunology Branch, National Institutes of Health, National Institute of Neurological Disorders and Stroke, 9000 Rockville Pike, Building 10, Room 5C-103, Bethesda, MD, 20892, USA.
Giovanna S
GS
Brunetto
Raya
R
Massoud
Emily C
EC
Leibovitch
Breanna
B
Caruso
Kory
K
Johnson
Joan
J
Ohayon
Kaylan
K
Fenton
Irene
I
Cortese
Steven
S
Jacobson
D000328:Adult; D000368:Aged; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D015490:HTLV-I Infections / Q000097:blood* / Q000134:cerebrospinal fluid* / Q000821:virology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D015493:Paraparesis, Tropical Spastic / Q000097:blood / Q000134:cerebrospinal fluid / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D019562:Viral Load
D004279:DNA, Viral