Polymerase chain reaction-restriction fragment length polymorphism method for detection of Calreticulin type-1 and type-2 mutations in myeloproliferative neoplasm

Calreticulin (CALR) mutations are included in the WHO diagnostic criteria of Myeloproliferative Neoplasm (MPN). Presence CALR mutation, aids their diagnosis by excluding reactive causes. There several molecular techniques to detect mutations; however, these require advanced equipment and technical expertise. An in-house “polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP)” method, using restriction enzymes MseI MluCI for detection type-1 type-2 mutations, was developed. PCR-RFLP method validated comparing its results with real-time PCR (RQ-PCR) CE-IVD marked Ipsogen® RGQ (Qiagen, Germany) kit. All positive samples MPN (n=26) were confirmed RQ-PCR, all negative (n=21) be negative, showing 100% sensitivity specificity mutation PCR-RFLP. The developed can used only a machine gel electrophoresis system. It is an inexpensive easily applicable alternative which adopted even small laboratory mutations.