Clinical application of catalytically cleavable fluorescence probe technology for multiplexing quantification of BCR–ABL1 fusion transcripts

• Our assay could be a reliable tool for the quantification of BCR-ABL1 transcripts.
• This is the first report applied CataCleave technique for human RNA quantification.
• CataCleave technology can be reliably used for quantification of human RNA.

BackgroundAccurate measurement of BCR–ABL1 fusion transcripts is critical for therapeutic stratification in patients with chronic myelogenous leukemia (CML). Previous studies have reported the variable performance of the existing quantitative reverse transcription polymerase chain reaction (RQ-PCR). Here, we developed a one-step multiplex RQ-PCR method based on the catalytically cleavable fluorescence probe technology for quantification of BCR–ABL1 transcripts.MethodsPerformance was evaluated with respect to the limit of detection (LoD), linearity, precision, and comparison on the VIIA7 Real-Time PCR system. Multiplex RQ-PCR was performed by the one-step and one-well reaction without the hands-on time.ResultsOur assay showed a LoD of 1.5 pg with linearity in the range of more than 4 logs of dilution. Intraassay, interassay, and total percent CVs at the concentration of 150 ng were 12.8%, 22.6%, and 28.0%, respectively. The assay correlated well with Asuragen's BCR/ABL1 Quant™ kit over a 6 log concentration range (r = 0.9967).ConclusionOur assay demonstrated comparable performance characteristics in comparison with previous RQ-PCR based on the TaqMan probe technology. We conclude that our method could be a reliable tool in the clinical setting.