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 mu g 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 P-32- or alkaline phosphatase-labelled SKI 12 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. (c) 2006 Elsevier B.V. All rights reserved.