RESUMO
Nested PCR (NPCR), a two-step procedure in which the products of a first PCR using 'outer' primers are reamplified using 'inner primers', has been successfully used to test for the chronic myeloid leukemia (CML)-specific bcr-abl transcripts. A major drawback of the conventional nesting strategy is linked to the opening of the reaction tube between the two successive PCR reactions, giving a risk of contaminating the second mix with amplicons. In this paper, the application of a new protocol for NPCR without reopening the reaction tube between the two steps of the procedure is described for the research of residual leukemic cells in the peripheral blood of 14 CML patients treated by bone marrow transplantation (BMT) or interferon (IFN). This assay which is both highly specific and sensitive, offers several advantages over the use of conventional NPCR: it is more sensitive, faster and decreases the risk of false-positive results. In addition, chemiluminescent detection of amplified DNA after transfer onto a nylon membrane, although comparable with radioactive hybridization in terms of sensitivity and speed, is more advantageous in safety and convenience. In conclusion, this assay could be adapted to a number of clinical diagnostic uses.
