Real-time quantitative PCR using hairpin-shaped clone-specific primers for minimal residual disease assessment in an animal model of human non-Hodgkin lymphoma

A multitude of molecular techniques for monitoring minimal residual disease in lymphoproliferative disorders have been described to date. Real-Time Quantitative PCR targeting Immunoglobulin Heavy chain patient-specific sequences is increasingly being used for molecular detection of residual neoplastic B-cells using allele-specific oligos. The establishment of individually tailored PCR assays with the extensive use of patient-specific fluorescent-labeled oligos may be cumbersome and expensive. The present study was aimed at evaluating the usefulness of recently described hairpin-shaped allele-specific primers, originally intended for typing single-nucleotide polymorphisms, for the assessment of minimal residual disease using SYBR Green intercalating dye. Three cloned and 2 sequenced clonogenic Ig heavy chain rearranged gene loci, obtained from 5 cases of canine spontaneous B-cell lymphoma, were used as an experimental model. Both standard linear and hairpin-shaped forward and reverse clone-specific primers were evaluated in terms of specificity, sensitivity and PCR efficiency. Hairpin-shaped primers were demonstrated to have achieved accurate results more consistently than the respective linear primers allowing the specific and sensitive quantification of minimal residual disease of lymphoproliferative disorders with fewer validation procedures and more flexibility on the assay design.