Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.
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 (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method.


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 HP(del) 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 HP(del) 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.


CONCLUSIONS
The detection rate of the HP(del) 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 HP(del).
Soejima
Mikiko
M
Department of Forensic Medicine and Human Genetics, Kurume University Hospital, Kurume, Japan.
Tsuchiya
Yuji
Y
Egashira
Kouichi
K
Kawano
Hiroyuki
H
Sagawa
Kimitaka
K
Koda
Yoshiro
Y
D000707:Anaphylaxis / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000517:prevention & control; D001323:Autoantibodies / Q000097:blood / Q000276:immunology; D052160:Benzothiazoles; D003959:Diamines; D005260:Female; D017353:Gene Deletion; D006242:Haptoglobins / Q000235:genetics*; D006801:Humans; D008297:Male; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines
D001323:Autoantibodies; D052160:Benzothiazoles; D003959:Diamines; D006242:Haptoglobins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I