Rapid detection of immunoglobulin heavy chain gene rearrangement by PCR and melting curve analysis using combined FR2 and FR3 primers

Abstract

Background: Immunoglobulin heavy chain (IgH) gene rearrangement test is a standard tool in diagnosing B-cell lymphoma. The BIOMED-2 multiplex PCR protocol has become the most commonly used laboratory method for detecting clonal IgH gene rearrangement. However, post-PCR procedure requires manual transfer of PCR product for analysis and is time-consuming. A novel strategy using LightCycler to continuously monitor fluorescence during melting curve analysis (MCA) can overcome these shortcomings. The previous studies published on this method were all restricted to FR3 primers of BIOMED-2. Methods: Real-time PCR and subsequent MCA were performed on 71 clinical DNA samples from formalin-fixed, paraffin-embedded tissues, including 40 with B-cell non-Hodgkin lymphomas and 31 with reactive lymphoid hyperplasia. We optimized the current method using FR3 primers and applied FR2 primers for the first time into MCA to detect IgH gene rearrangement. Polyacrylamide gel electrophoresis and capillary gel electrophoresis were also performed on all lymphoma samples with the identical FR2 primers. Results: MCA of combined FR2 and FR3 primer sets yielded the sensitivity and the specificity equal to 70 % (28/40) and 100 % (31/31), respectively. Addition of FR2 primers increased the sensitivity by 12.5 % (5/40) comparing to FR3 primers alone. MCA was slightly more sensitive than polyacrylamide gel electrophoresis and comparable to capillary gel electrophoresis to detect clonal IgH gene rearrangement. Conclusions: Combined PCR and DNA melting curve analysis in a closed system can reduce cross-contamination risk. This method can test 96 samples simultaneously within 90 min and therefore, it is high-throughput and faster. PCR-MCA in the LightCycler system has potential for evaluating monoclonal IgH gene rearrangement in a clinical environment.