Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials

作者：Soejima Mikiko; Tsuchiya Yuji; Egashira Kouichi; Kawano Hiroyuki; Sagawa Kimitaka; Koda Yoshiro^*

来源：Transfusion, 2010, 50(6): 1322-1327.

DOI：10.1111/j.1537-2995.2009.02581.x

BACKGROUND:
Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HPdel) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HPdel before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method.
STUDY DESIGN AND METHODS:
A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HPdel zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates.
RESULTS:
Allelic discrimination of HPdel yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method.
CONCLUSION:
The detection rate of the HPdel allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HPdel.

• 出版日期2010-6