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Detection of the MYD 88 mutation by the combination of the allele‐specific PCR and quenching probe methods
Author(s) -
Nogami S.,
KawaguchiIhara N.,
Shiratori E.,
Ohtaka M.,
Itoh M.,
Tohda S.
Publication year - 2017
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/ijlh.12598
Subject(s) - microbiology and biotechnology , allele , mutant , missense mutation , restriction fragment length polymorphism , high resolution melt , melting curve analysis , detection limit , wild type , mutation , variants of pcr , biology , polymerase chain reaction , chemistry , genetics , gene , chromatography
Summary Introduction The MYD 88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele‐specific PCR ( AS ‐ PCR ), PCR ‐restriction fragment length polymorphism ( PCR ‐ RFLP ), and high‐resolution melting analysis ( HRM ) are currently used to detect the mutation; however, they are either time‐consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe ( QP ) technique and AS ‐ PCR . Method A lymphoma cell line heterozygous for the MYD 88 mutation, two wild‐type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS ‐ PCR , PCR ‐ RFLP , HRM , and QP , and their detection sensitivity was examined using the mixtures of the mutant and wild‐type DNA . Results For mutation‐carrying heterozygous samples, the QP method produced W‐shaped melting profiles presenting curves derived from the wild‐type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS ‐ PCR and QP ( AS ‐ QP ) improved the sensitivity to 0.62% of the mutant allele. Conclusion The AS ‐ QP analysis is rapid and minimally improves detection sensitivity compared to the AS ‐ PCR .