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

Eun Hae Cho; Sang Gon Lee; Jeong Ho Seok; Bo Ya Na Park; Eun Hee Lee

doi:10.3343/kjlm.2009.29.6.589

Journal List > Korean J Lab Med > v.29(6) > 1011603

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Cho, Lee, Seok, Park, and Lee: Evaluation of Two Commercial HLA-B27 Real-Time PCR Kits

Original Article

Korean J Leg Med 2009;29(6):589-593.

Published online: 14 January 2009

DOI: https://doi.org/10.3343/kjlm.2009.29.6.589

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

Eun Hae Cho, M.D., Sang Gon Lee, M.D., Jeong Ho Seok, M.D., Bo Ya Na Park, M.S., Eun Hee Lee, M.D.

Greencross Reference Laboratory, Yongin, Korea

Corresponding author: Eun Hae Cho, M.D. Greencross Reference Laboratory, 314 Bojeong-dong, Giheung-gu, Yongin 449-913, Korea Tel: +82-31-260-9216, Fax: +82-31-260-9638 E-mail: ssea74@yahoo.co.kr

Revised 13 March 200923 July 2009 Accepted 8 September 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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^® HLA-B27 real-time PCR kit (Bioneer, Korea) with TaqMan probes and the Real-Q™ 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 realtime 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.

Keywords: Real Time PCR, HLA-B27, PCR-SSP

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Fig. 1.

Histogram representation showing the distribution of C_t FAM (HLA B-27) and C_t TAMRA (GAPDH) from 61 samples on genotyping by Taqman allele-specific amplification. All samples had C_t TAMRA values <26, and 28 positive samples had C_t FAM values <28.

Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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.

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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