Korean J Lab Med.  2009 Dec;29(6):589-593. 10.3343/kjlm.2009.29.6.589.

Evaluation of Two Commercial HLA-B27 Real-Time PCR Kits

• Cho EH
• Lee SG
• Seok JH
• Park BY
• Lee EH

• KMID: 1781615
• DOI: http://doi.org/10.3343/kjlm.2009.29.6.589

Abstract

BACKGROUND
The standard PCR with sequence-specific primers (SSP) is a widely used method of HLA-B27 typing in clinical practice. The aim of our study was to evaluate 2 Korean HLA-B27 kits with different real-time PCR chemistries. METHODS: To validate the accuracy of real-time PCR kits, we selected 28 HLA-B27-positive samples and 33 HLA-B27-negative samples with a wide range of different HLA-B specificities typed by standard PCR-SSP. The 2 real-time PCR kits used were the AccuPower(R) HLA-B27 real-time PCR kit (Bioneer, Korea) with TaqMan probes and the Real-Q(TM) HLA-B*27 detection kit (BioSewoom, Korea) with SYBR Green I dye for melting curve analysis. RESULTS: All 61 samples typed by PCR-SSP demonstrated a perfect concordance with the 2 real-time PCR assays. It was possible to clearly discriminate between HLA-B27-positive and -negative samples in both real-time assays. CONCLUSIONS: In summary, both real-time PCR assays for HLA-B27 were fast, reliable, well-adapted for routine laboratory testing, and attractive alternatives to the conventional PCR-SSP method.

Keyword

Real Time PCR; HLA-B27; PCR-SSP

MeSH Terms

HLA-B27 Antigen/*analysis
Histocompatibility Testing/*methods
Humans
Organic Chemicals/chemistry
Polymerase Chain Reaction
Reagent Kits, Diagnostic
Transition Temperature

Figure

• 

  Fig. 1. Histogram representation showing the distribution of Ct FAM (HLA B-27) and Ct TAMRA (GAPDH) from 61 samples on genotyping by Taqman allele-specific amplification. All samples had Ct TAMRA values <26, and 28 positive samples had Ct FAM values <28. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  Fig. 2. Fluorescence signal versus cycle number plot during the amplification of 2 HLA-B27 positive cases out of six different human DNA samples with primers specific for HLA-B27 (green signal) and GAPDH (yellow signal). Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  Fig. 3. Melting peaks specific for HLA-B27 around 86.5°C and β-globin around 83.8°C during the melting curve analysis of 2 HLA-B27 positive cases out of 6 different human DNA samples with SYBR Green I dye.

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