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 of CALR mutation, aids in their diagnosis by excluding reactive causes. There are several molecular techniques to detect CALR 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 and MluCI for detection of CALR type-1 and type-2 mutations, was developed. PCR-RFLP method was validated by comparing its results with real-time PCR (RQ-PCR) using CE-IVD marked Ipsogen® CALR RGQ PCR (Qiagen, Germany) kit. All PCR-RFLP positive CALR type-1 and type-2 mutations samples of MPN (n=26) were confirmed positive by RQ-PCR, and all negative samples (n=21) were confirmed to be negative, showing 100% diagnostic sensitivity and specificity for CALR mutation detection by PCR-RFLP. The developed PCR-RFLP method can be used for detection of CALR type-1 and type-2 mutations with only a PCR machine and gel electrophoresis system. It is an inexpensive and easily applicable alternative method which can be adopted even by a small molecular laboratory for detection of CALR mutations.