Non-radioisotope detection of pol sequences of HTLV-1 proviral DNA: Standardisation and sensitivity analysis.
Proviral DNA amplification methods may be used for identification of HTLV-1 infection or in basic virology research. Published standardised methods in this regard usually depend on hybridisation of PCR products with radioisotope-labelled probes. However, this procedure has limited use in routine testing, due to environmental and health risks. The aim was to assess the feasibility of routine use and the accuracy of an alternative detection system that employs an HTLV-1-specific enzyme-labelled probe. For this purpose DNA was extracted from MT-2 cells, quantified and submitted to serial dilution (1:10), starting from 1.2 microg of genomic DNA. Primary and nested PCR amplifications of pol sequences of the HTLV-1 genome were carried out with standardised primers (SK110/111 and POL1.1/3.1). After Southern blotting, two different detection systems were compared, consisting of hybridisation with either 32P- or alkaline phosphatase-labelled SK112 probes. Both detection systems yielded similar results, detecting PCR products generated from 120 pg of DNA (genomic DNA amount equivalent to 20 diploid human cells) after primary and nested PCR. The alkaline phosphatase-labelled detection technique was feasible for the diagnosis of HTLV-1 with the advantage of precluding the handling of radioisotopes.
Costa
José Marcos Pereira
JM
Virology Laboratory (LIM-52), Department of Infectious Diseases, School of Medicine, University of São Paulo, Av. Dr. Eneas de Carvalho Aguiar 470, 05403-000 São Paulo, SP, Brazil.
Sumita
Laura Massami
LM
Segurado
Aluisio Cotrim
AC
D000469:Alkaline Phosphatase / Q000032:analysis; D001345:Autoradiography; D015139:Blotting, Southern; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D015751:Genes, pol; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010761:Phosphorus Radioisotopes / Q000032:analysis; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D000469:Alkaline Phosphatase