Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma.
Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.
Martinez-Lopez
Joaquin
J
Servicio de Hematología, Hospital Universitario, 12 de Octubre. Av. de Cordoba, s/n Madrid 28041, Spain. mpmartinezsa@yahoo.es
Martinez-Sanchez
Pilar
P
Garcia-Sanz
Ramon
R
Sarasquete
Maria Eugenia
ME
Ayala
Rosa
R
Gonzalez
Marcos
M
Bautista
Jose Manuel
JM
Gonzalez
David
D
San Miguel
Jesus
J
Garcia-Effron
Guillermo
G
Lahuerta
Juan Jose
JJ
D001483:Base Sequence; D003199:Computer Systems; D032921:Consensus; D015342:DNA Probes; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000175:diagnosis* / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D017434:Protein Structure, Tertiary / Q000235:genetics
D015342:DNA Probes; D007143:Immunoglobulin Heavy Chains