Rapid HLA-DR fluorotyping based on melting curve analysis.
Real-time polymerase chain reaction (PCR) has been used in the study of human leukocyte antigen (HLA) genotyping as a potential alternative for routinely used molecular methods such as PCR-sequence specific primers (PCR-SSP) and PCR-sequence specific oligonucleotide probes (PCR-SSO). Combined with fluorescent dye like SYBR GREEN I, it has more advantages such as low cost and consistent background. The aim of this study was to optimize the fluorescent dye-based method and introduce it into the fluorotyping for HLA-DR lotus. 24 pairs of allele-specific primers and 1 pair of internal control were optimized to discriminate HLA-DRB1, -DRB3/B4/B5 alleles. Additionally, conditions of real-time PCR amplifying and melting curve recording had been improved for convenient and clear readout. Forty-two clinical samples previously typed by conventional PCR-SSP or sequence based typing (SBT) were tested and all got identical results. With this technique, 15 DNA samples can be assayed in parallel within 2 hours on the Real-time PCR instrument. These data strongly suggest a rapid HLA-DR fluorotyping method based on melting curve analysis, which could be a more economic and automatic alternative for clinical HLA-DR typing.
Liu
Yang
Y
Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
Zhu
Qiyun
Q
Zhu
Naishuo
N
D000483:Alleles; D052160:Benzothiazoles; D003959:Diamines; D006684:HLA-DR Antigens / Q000276:immunology*; D006650:Histocompatibility Testing; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D052160:Benzothiazoles; D003959:Diamines; D006684:HLA-DR Antigens; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I