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Rapid detection of haptoglobin gene deletion in alkaline-denatured blood by loop-mediated isothermal amplification reaction.

Abstract
Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin antibodies. Being homozygous for the haptoglobin gene deletion allele (HP(del)) is the only known cause of congenital anhaptoglobinemia, and detection of HP(del) before transfusion is important to prevent anaphylactic shock. In this study, we developed a loop-mediated isothermal amplification (LAMP)-based screening for HP(del). Optimal primer sets and temperature for LAMP were selected for HP(del) and the 5' region of the HP using genomic DNA as a template. Then, the effects of diluent and boiling on LAMP amplification were examined using whole blood as a template. Blood samples diluted 1:100 with 50 mmol/L NaOH without boiling gave optimal results as well as those diluted 1:2 with water followed by boiling. The results from 100 blood samples were fully concordant with those obtained by real-time PCR methods. Detection of the HP(del) allele by LAMP using alkaline-denatured blood samples is rapid, simple, accurate, and cost effective, and is readily applicable in various clinical settings because this method requires only basic instruments. In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests.
AuthorsMikiko Soejima, Kouichi Egashira, Hiroyuki Kawano, Atsushi Kawaguchi, Kimitaka Sagawa, Yoshiro Koda
JournalThe Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 13 Issue 3 Pg. 334-9 (May 2011) ISSN: 1943-7811 [Electronic] United States
PMID21497293 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
Chemical References
  • Haptoglobins
Topics
  • Base Sequence
  • Gene Deletion
  • Gene Order
  • Haptoglobins (genetics)
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques
  • Sensitivity and Specificity

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