Rapid and high-resolution detection of IgH gene rearrangements using PCR and melting curve analysis.
The analytical methods of Southern blot hybridization (SBH) and the polymerase chain reaction (PCR) for complementarity determining region-3 (CDR3) are fundamental for detecting IgH gene rearrangement. However, there are problems stemming from the characteristics of both methods; especially, the long turn around time (TAT) because of the complex process in the SBH, and the low analytical sensitivity for amplicons in the PCR. Thus, to improve the PCR procedure, we investigated the application of detecting the clonal amplicons based on the different melting Temperature (T(m)) in internal melting domains corresponding to the CDR3 hypervariable region. Our new protocol is based on the combination of a LightCycler Technology with high-speed amplification, and Idaho-Technology with rapid and high-resolution melting curve analysis (MCA), designated PCR-MCA. This method can provide the results within 3 h with an analytical sensitivity of 10(-3). The diagnostic sensitivity and specificity relative to the results documented with the SBH analysis were 89.2% and 100%, respectively. This indicates that the new protocol of PCR-MCA is acceptable for clinical testing; especially, PCR-MCA is relevant in terms of the rapid and sensitive detection of IgH clonality within amplicons.
Uemura
A
A
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, Nagasaki City, Nagasaki, Japan.
Mori
S
S
Sugahara
K
K
Akamatsu
N
N
Tsuruda
K
K
Tsukasaki
K
K
Hirakata
Y
Y
Atogami
S
S
Hasegawa
H
H
Yamada
Y
Y
Kamihira
Shimeru
S
D015139:Blotting, Southern / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007938:Leukemia / Q000235:genetics; D008232:Lymphoproliferative Disorders / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D007143:Immunoglobulin Heavy Chains