Rapid HLA-DR Fluorotyping Based on Melting Curve Analysis

Abstract

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.

A rapid simple approach to quantify chromosome conformation capture

“…MCA is used widely to discriminate qualitatively between multiple variants, for example in genotyping and microbial detection (28,30,42). Here we used MCA as both a qualitative and quantitative tool.…”

High-throughput sequence-based typing strategy for HLA-DRB1 based on real-time polymerase chain reaction

“…Normally high-resolution HLA genotyping requires haplotype separation for the reduction of ambiguities caused by the cis/trans difficulty and can be achieved by group-specific PCR [4] and other methods [5]. During the last decade real-time PCR had become available, and has proved to be a convenient approach for low-to medium-resolution HLA typing using the advantage of melting curve analysis based on hybridization probes [6] and SYBR Green chemistry [7].…”