Fluorescent and colorimetric RT-LAMP as a rapid and specific qualitative method for chronic myeloid leukemia diagnosis

Abstract

The detection of BCR-ABL1 mRNA transcripts is essential to molecular chronic myeloid leukemia (CML) diagnosis. In most cases, the RT-qPCR technique is performed as the gold standard diagnosis tool for clinical cases. However, this method requires expensive reagents and equipment, such as a real-time thermal cycler, probes and master mix. Consequently, the development and validation of simple and lowcost methods are essential for a rapid CML diagnosis in less specialized and equipped centers. In this study, we develop and demonstrate an accessible, rapid, and low-cost method using RT-LAMP for BCR-ABL1 detection in both cell lines and CML clinical samples, using colorimetric and uorescent assays. Differently to the Q-LAMP assay described in 2019 by Stella and collaborators, the samples here were analyzed by RT-qPCR and the results were compared to the results obtained by uorescent and colorimetric RT-LAMP. The obtained data indicates that the proposed method here described is a cheaper, robust and speci c approach for CML diagnosis with outstanding performance.

CATCH: high specific transcriptome-focused fusion gene variants discrimination

“…S5 and Table S3, ESI†). 14,21–28 Furthermore, among the mixed samples with different concentrations of RNA targets, we observed that the LOD of the samples containing EML4-ALK V1 in 10 ng total RNA was 0.5% (Fig. 2D).…”

“…Fig.2A-Cshowed a good linear relationship between the peak of fluorescence spectra and input concentrations (R 2 = 0.993). The proposed CATCH approach enabled EML4-ALK V1 detection in the femtogram range, which has been improved by four orders of magnitude compared with the CATCH assay without subsequent CHA amplification, and also had a better analytical performance than other methods (Fig.S5and TableS3, ESI †) 14,[21][22][23][24][25][26][27][28]. Furthermore, among the mixed samples with different concentrations of RNA targets, we observed that the LOD of the samples containing EML4-ALK V1 in 10 ng total RNA was 0.5% (Fig.2D).We also evaluated the detection capability of the proposed approach by a standard recovery experiment and repeatability study.…”