Real-time quantitative PCR for rapid detection of translocations, amplifications, polymorphisms, and viral/bacterial load in blood or formalin-fixed paraffin embedded tissues

B9 The Unite d9Interface clinique of the Hotel-Dieu de Quebec Research Center is a laboratory specialized in cellular and molecular diagnostic. We offer an array of in-house diagnostic assays to the scientific and medical communities for the detection of translocations involved in leukemia, lymphoma and sarcoma, detection of monoclonality in T cell and B cell malignancies, detection of oncogene amplification, genotyping of polymorphisms, and quantification of virus and bacteria. In order to offer faster and more precise molecular diagnostic results, we have developed many qPCR assays and have evaluated their accuracy and sensitivity as compared to the method previously used. We have compared Her2/neu gene amplification detection using qPCR, FISH and IHC in formalin-fixed paraffin embedded tissues (FFPE) tissues, and have found an excellent concordance (96%) between qPCR and FISH results, while it was only 83% between FISH and IHC, and 90% for FISH performed in two different laboratories. The sensitivity of BCR-ABL translocation detection has been measured for RT-qPCR, RT-PCR and nested RT-PCR in chronic myelogenous leukemia patients. RT-qPCR could detect ratio of BCR-ABL/G6PDH as low as 0.003%, which was more sensitive than RT-PCR and slightly less than nested RT-PCR, although only RT-qPCR allows ratio quantification to monitor disease progression. Hybridization probes and qPCR are used to genotype a variety of polymorphisms, including JAK2 V617F mutation involved in myeloproliferative disorders. This genotyping method is safe and simple and does not require any subsequent step such as RFLP or sequencing. Synovial sarcoma is characterized by the translocation t(x;18) which involves the SYT gene with the SSX1 or the SSX2 gene; both rearrangement can be detected and distinguished in the same RT-qPCR reaction without electrophoresis of PCR products. Using hybridization probes and qPCR, we have developed a sensitive method to detect Mycobacterium tuberculosis in FFPE tissues and to simultaneously distinguish tuberculosis from other mycobacterium strains. A similar method was used to quantify polyomavirus (BK and JC) in the blood or urine of renal transplant recipients, allowing to monitor nephropathy risks without biopsies. Our tests have shown that qPCR is equally or more sensitive than other methods to detect translocations, amplifications, polymorphisms, and viral/bacterial load. In addition, the use of hybridization probes can allow simultaneous identification or genotyping without the need to perform another step, such as culture, electrophoresis or sequencing. qPCR can be a good choice to develop rapid, efficient and precise assays for molecular diagnostic and personalized therapy.