Automated JAK2V617F quantification using a magnetic filtration system and sequence-specific primer-single molecule fluorescence detection.

We established an automated mutational analysis detection system using magnetic filtration and the sequence-specific primer-single molecule fluorescence detection (SSP-SMFD) assay to identify the janus activating kinase-2 (JAK2)(V617F). DNA was extracted from 100 microL of whole blood automatically by a magnetic filtration system. The JAK2 1849G-->T mutation occurs in chronic myeloproliferative disorder (CMPD), and the detection of this change has diagnostic potential. To detect and semiquantitate this mutation, we used two artificial oligonucleotides (wild-type specific and mutated-type specific) and performed the SSP-SMFD assay using an automated fluorescence cell sorter measuring device. The SSP-SMFD assay can detect the presence of a minimum of 5% of the mutated artificial oligonucleotide, thus indicating that this technique is available in detecting contamination of at least 5% cells with the homozygous JAK2(V617F) mutation. Based on this technique, we analyzed 94 patients with CMPD and compared with the results obtained by the polymerase chain reaction (PCR)-direct sequence. Two homozygous JAK2(V617F) patients were identified as heterozygous JAK2(V617F) by the PCR-direct sequence, and four patients judged as wild-type JAK2 by the PCR-direct sequence were identified as heterozygous JAK2(V617F) by the SSP-SMFD method. Our automated system is simple and suitable for high-throughput analysis in detecting JAK2(V617F) with a threshold detection limit of 5%.