Development and clinical evaluation of oligonucleotide microarray for HLA-AB genotyping.
BACKGROUND
The antigens encoded by human leukocyte antigen (HLA) genes are primary antigens in immunological response of transplantation, and genotypes of HLA-A, B and DRB1 must be determined on donors and recipients before the transplantation is carried out. In this study, oligonucleotide microarray for HLA-AB genotyping was prepared and evaluated.


METHODS
Oligonucleotide probes were designed based on the sequence of the different genotypes of HLA-A and B and were fixed on silylated glass slides to form a microarray. Fluorescence-labeled target DNA was obtained by asymmetric polymerase chain reaction amplification of exon 2 and exon 3 of HLA-A and -B genes and hybridized to the microarray. The hybridized microarray was subsequently scanned and the result was analyzed by software in order to determine the genotypes of the tested sample.


RESULTS
The sensitivity of the microarray for HLA-AB genotyping was 10 ng/microl, with a coincidence rate of 100% with international reference, and a combined variation value of detected signal below 15%. Analysis of genotyping of 1295 cases of clinical samples showed that the general coincidence rate between the microarray method and conventional method (polymerase chain reaction-sequence-specific primer and flow cytometry reverse polymerase chain reaction sequence-specific oligonucleotide) was HLA-A: 99.61% and HLA-B: 98.30%, respectively. A total of five out of seven samples that had conflicting results of genotypes were proved to be microarray-assay reliable by DNA sequencing, suggesting a higher accuracy of the microarray method.


CONCLUSIONS
The microarray for HLA-AB genotyping is satisfactory for clinical use in HLA-AB genotyping with its good specificity, sensitivity and reproducibility.
Zhang
Fan
F
Beijing Institute of Radiation Medicine, Beijing 100850, China.
Hu
Shouwang
S
Huang
Jian
J
Wang
Hui
H
Wen
Zheng
Z
Yongyao
Geng
G
Wang
Shengqi
S
D015342:DNA Probes; D004311:Double-Blind Method; D005091:Exons / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry; D005838:Genotype; D015234:HLA-A Antigens / Q000276:immunology*; D015235:HLA-B Antigens / Q000276:immunology; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software
D015342:DNA Probes; D015234:HLA-A Antigens; D015235:HLA-B Antigens; D009841:Oligonucleotides