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. The absence of a serum protein such as IgA or haptoglobin (Hp) is one of the factors that can lead to anaphylactic transfusion reactions due to production of serum antibodies against the absent protein after a transfusion. 1 At present, a homozygous deletion of the haptoglobin gene (HP del ) is the only known cause of anhaptoglobinemia.Human Hp has a genetic polymorphism of two codominant alleles, HP 1 and HP 2 , that give rise to the three common phenotypes, Hp1, Hp2-1, and Hp2. 2,3 The HP 2 allele appears to have occurred by a 1.7-kb intragenic duplication of exons 3 and 4 of the HP 1 allele ( Figure 1A). Anomalous inheritance of the Hp phenotypes was encountered during determinations of parentage, and HP del was identified by genetic analyses of one such family in Japan. 4 The HP del allele lacks an approximately 28-kb segment of chromosome 16 extending from the promoter region of the HP gene to intron 4 of the haptoglobinrelated gene (HPR) ( Figure 1A). 4,5 The HP del allele has been found only in East and Southeast Asian populations (Chinese, Korean, Japanese, Mongols, Thais, and Indonesians), not in African, West and South Asian, and European populations so far. [5][6][7][8][9] Detection of homozygosity for HP del before blood transfusion or blood component infusion is important to prevent severe side effects of transfusion because washed red blood cells and platelet concentrate do not cause transfusion-related anaphylactic reactions. 10 Recently, we established two real-time PCR methods for detection of HP del by use of a 5=-nuclease assay using dual-labeled (TaqMan; Applied Biosystems, Foster City, CA) probes and SYBR Green I (Invitrogen, Carlsbad, CA). 11,12 These methods are rapid, robust, and suitable for high-throughput analysis but req...

Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings

“…Similar methods have been used prior to loop-mediated isothermal amplification. 35 Incubation at alkaline pH rapidly disrupts cell and nucleus membranes, denatures nucleases, and dissolves DNA. A 1:100 dilution step is necessary following lysis to dilute the components of whole blood that inhibit RPA to levels tolerated by the assay, while still maintaining a high enough concentration of DNA to fall within the dynamic range of the assay.…”

Static self-directed sample dispensing into a series of reaction wells on a microfluidic card for parallel genetic detection of microbial pathogens

“…Ultimately, decentralized genetic testing must also allow for sample-in-results-out, with automation, minimization, or elimination of sample preparation (DNA extraction/purification). LAMP and direct amplification have been previously demonstrated with many different targets (bacterial, viral, human, fungal), and sample types with varied concentrations in the amplification reaction including 1 to 50 % blood (Curtis et al 2009 ; Ebbinghaus et al 2012 ; Masaomi et al 2003 ; Patterson et al 2013 ; Soejima et al 2011 ), 10 % stool, (Francois et al 2011 ), 20 % urine samples (Hill et al 2008 ; Koizumi et al 2012 ), and 32 % nasal swabs (Nie et al 2012 ). As such, the airlock cards were tested with these sample types at varying sample concentrations in the final LAMP reaction mixture.…”

Advances in Directly Amplifying Nucleic Acids from Complex Samples

“…Simple treatments with heat [59,[65][66][67][68][69][70][71][72][73][74][75][76] or chemicals [73,[77][78][79][80][81] can increase the sensitivity of the LAMP or RT-LAMP reaction. Most impressive are the examples of direct amplification of DNA in blood with LAMP-based technologies [82,83] and other isothermal amplification methods like MDA [84].…”