Detection of the MYD88 mutation by the combination of the allele‐specific PCR and quenching probe methods. (1st January 2017)

• Record Type: Journal Article
• Authors: Nogami, S.; Kawaguchi‐Ihara, N.; Shiratori, E.; Ohtaka, M.; Itoh, M.; Tohda, S.
• Is Part Of: International journal of laboratory hematology. Volume 39:Number 2(2017:Apr.)
• Page Start: 163
• Page End: 168
• Publication Date: 2017-01-01
• Subjects: MYD88 -- quenching probe -- allele‐specific PCR -- high‐resolution melting analysis -- Waldenström's macroglobulinemia; Hematology -- Periodicals; Blood -- Diseases -- Periodicals; Hematology -- Periodicals; 616.15005
• DOI: 10.1111/ijlh.12598
• Languages: English
• ISSNs: 1751-5521

Abstract

Summary: Introduction: The MYD88 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 MYD88 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.