Ultra Rapid, One Step Molecular Detection of BCR-ABL Major and Minor Transcripts by Isothermal RT-Loop Mediated Amplification Reaction.

The molecular identification of the BCRABL transcripts in clinical samples is actually based on a conventional Reverse Transcription Polymerase Chain reaction (RT-PCR). Here we present a novel molecular method, based on Loop Mediated Amplification assay that, starting from RNA (RT-LAMP) in one single tube reaction ensures a rapid and simultaneous detection of either the BCR-ABL p190 or p210 fusion transcript as well as the housekeeping gene used as internal quality control.

The BCR-ABL RT-LAMP is a multiplex, isothermal method for retro-transcription, amplification and detection of the Minor (p190) and Major (p210) t(9;22) transcripts and the endogenous Gusb RNA, as internal control for validation of negative results. The employment of fluorescent specific probes allows real-time monitoring of the reaction, so that the test result is obtained in a single, homogeneous step. RT-LAMP is carried out on the Liaison IAM, a 8-wells manageable instrument suitable for isothermal reactions. Liaison IAM incubates at constant temperature, monitors the fluorescent signals and the data produced can be analyzed, upon connection to up to 6 other instruments, for a throughput of 8 to 48 samples. Thanks to the three channels of fluorescence, it can monitor multiplex assays, providing elaborated final objective results with no need of further data analysis by the operator.

The level of sensitivity of the triplex BCR-ABL RT-LAMP has been analytically evaluated directly on serial dilutions of RNA extracted respectively from t(9;22) positive cell lines (TOM-1 for p190 or K-562 for p210) into wild type RNA from HL-60 cell line (30 replicates). The p190 and p210 transcripts have been detected and distinguished down to 10⁻⁴ and 10⁻⁵respectively within 50 minutes. The assay demonstrated 100% specificity since 70 replicates of wild type RNA from 7 cell lines resulted BCR-ABL negative and GUSb positive (internal amplification control). This assay was validated on 60 clinical samples (30 white blood cells RNA from Chronic Myeloid Leukemia, 30 mononuclear cells RNA from B-lineage Acute Lymphoblastic Leukemia). All these samples were obtained at diagnosis and were previously analyzed by conventional RT-PCR. RT-LAMP detected and identified the BCR-ABL fusion transcripts correctly in all cases with a 100% concordance with the reference method. Fully concordant results were obtained also on 30 RNA samples from patients affected by t(9;22) negative hematologic malignancies and on 30 RNA samples obtained from healthy donors in which the RT-LAMP amplified exclusively the housekeeping GUSb transcript.

The triplex p190-p210-GUSb RT-LAMP is a one-step procedure for specific, highly sensitive and rapid molecular detection of the BCR-ABL fusion transcripts. The semi-automatic instrument Liaison IAM, simplifies the entire procedure, reduces the contamination risks deriving from the conventional, multi step RT-PCR and significantly improve the diagnostic lab routine.

D'agostini:DIASORIN S.p.A: Employment. Minnucci:Diasorin S.p.A.: Employment. Amicarelli:DIASPRIN S.p.A.: Employment. Pultrone:DIASORIN S.p.A.: Employment. Tettamanzi:DIASORIN S.p.A.: Employment. Salmoiraghi:DIASORIN S.p.A.: Consultancy. Spinelli:DIASORIN S.p.A: Consultancy. Colotta:DIASORIN S.p.A: Employment. Rambaldi:DIASORIN S.p.A: Consultancy.