Establishment of scalable nanoliter digital LAMP technology for the quantitative detection of multiple myeloproliferative neoplasm molecular markers

Abstract

Myeloproliferative neoplasms (MPNs) are a type of chronic hematological tumor accompanied by bone marrow failure or leukemia. A nanoparticle-assisted digital loop-mediated isothermal amplification (nano-dLAMP) platform was established for the analysis of MPNs in this study. Microarray chips with four physical partitions were fabricated for the simultaneous detection of calreticulin type 1 (CALR-1), calreticulin type 2 (CALR-2), and janus kinase 2 V617F (JAK2 V617F) mutations and an internal reference gene. Polymerase chain reaction (PCR) additives and nanoparticles were used to make the traditional loop mediated isothermal amplification (LAMP) suitable for nanoliter-scale amplification. The results suggested that nanoparticles could improve the amplification performance of nanoliter LAMP. Quantitative detection of the main MPN molecular markers could be performed simultaneously in one four-partition microarray chip within 1 h. The detection sensitivity values for CALR-1, CALR-2, and JAK2 V617F were 0.5 %, 0.1 %, and 0.5 % mutation burden, respectively. The agreement between the developed platform and the commercial Quantstudio 3D was high at 99 % (280/281). This accurate, rapid, multiplex, and inexpensive nano-dLAMP platform could be a promising tool for clinical diagnosis in the future.