A triplex probe-based TaqMan qPCR assay for Calreticulin type I and II mutation detection.

Background: Calreticulin (CALR) exon 9 frameshift mutations have recently been identified in 30-40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) without JAK2 or MPL mutations. We aimed to develop a qPCR assay to screen type I and II mutations of CALR. Methods: Three different fluorescent-labeled hydrolysis probes and one pair of primers in a closed-tube system were developed to detect CALR type I and II mutations and distinguish them from wild-type. The sensitivity and specificity were validated using TA-cloning plasmids containing CALR wild-type and type I and II mutants, respectively. Fifty-nine ET and PMF specimens were screened by TaqMan qPCR and sequenced by Sanger sequencing. For intraassay validation, 20 replicates of the assay were performed with each sample. For inter-assay validation, four replications of each sample were carried out and repeated continuously for 5 days. Results:Wefound that triplex probe-based TaqManqPCR was reliable in detecting CALR type I and II mutants within DNA that was diluted to 1%of total DNA with the wild-type DNA as background. In 59 patient specimens, six of the observed mutations of CALR were type I and five were type II. Genotyping results obtained from TaqMan qPCRwere 100%concordantwith Sanger sequencing. The intra- and inter-assay CVs of TaqMan qPCR were less than 3%, respectively. Conclusions: Triplex probe-based TaqMan qPCR is an accurate and sensitive method for screening ET or PMF patients with type I and II mutations in CALR. [ABSTRACT FROM AUTHOR]