Development and validation of a real-time PCR assay for a novel HTLV-1 tax sequence detection and proviral load quantitation.
J Virol Methods
; 189(2): 383-7, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23499262
ABSTRACT
A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 105 to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×104 copies/106 PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Vírus Linfotrópico T Tipo 1 Humano
/
Infecções por HTLV-I
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Genes pX
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Provírus
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Carga Viral
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Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Virol Methods
Ano de publicação:
2013
Tipo de documento:
Article