Rapid HLA-DR fluorotyping based on melting curve analysis

Yang Liu, Qiyun Zhu, Naishuo Zhu

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)507-521
Number of pages15
JournalImmunological Investigations
Volume36
Issue number4
DOIs
StatePublished - Jul 2007

Keywords

  • Fluorotyping
  • Human leukocyte antigen
  • Melting curve
  • SYBR GREEN

ASJC Scopus subject areas

  • Immunology

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