A population-wide applicable HLA-DQ2 and DQ8 genotyping using DNA from dried blood spots and duplex allele-specific qPCR amplification.
Genotyping of HLA-DQ2 and DQ8 haplotypes is important for diagnosis or for screening of early risk detection of celiac disease or type 1 diabetes. Usually, venous blood DNA extraction and expensive and time consuming amplification are used, that hinder population-wide studies. We assayed a friendly HLA-DQ2 and DQ8 genotyping procedure using a combination of DNA from dried blood spot (DBS) and duplex allele-specific qPCR amplification using SYBR Green. DNA was extracted using home-made buffers and compared to an extraction commercial kit. Duplex reactions by qPCR were designed using each Tm allele amplicon for reference samples (positive HLA-DQ2 or DQ8) with allele-specific primers. DBS samples from 558 children (7.99 ± 2.47 y) were collected. The DNA final yield obtained by the home-made extractive procedure was higher than from the commercial kit (1.11 ± 0.56 vs 0.23 ± 0.14 μg), while the quality was similar for both DNA samples. There was concordance in the amplification profiles for DNA samples obtained with both methods. All of four alleles from DQ2 and DQ8 haplotypes were accurately identified in duplex reactions. By using DBS samples and DNA extraction home-made procedure, the costs were reduced by 60%. The whole procedure is cost-effective for HLA-DQ2 and DQ8 genotyping.
a Coordinación de Nutrición , Centro de Investigación en Alimentación y Desarrollo , Hermosillo , Sonora , México.
Sandra
S
Aguayo-Patrón
a Coordinación de Nutrición , Centro de Investigación en Alimentación y Desarrollo , Hermosillo , Sonora , México.
Lizbeth
L
Beltrán-Sauceda
a Coordinación de Nutrición , Centro de Investigación en Alimentación y Desarrollo , Hermosillo , Sonora , México.
Ana María
AM
Calderón de la Barca
D000483:Alleles; D052160:Benzothiazoles; D002446:Celiac Disease / Q000175:diagnosis / Q000235:genetics / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000235:genetics / Q000276:immunology; D003959:Diamines; D059788:Dried Blood Spot Testing / Q000379:methods*; D005260:Female; D005820:Genetic Testing; D060005:Genotyping Techniques / Q000379:methods*; D006683:HLA-DQ Antigens / Q000145:classification / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D006683:HLA-DQ Antigens; C053186:HLA-DQ2 antigen; C109756:HLA-DQ8 antigen; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA
Celiac disease; HLA-DQ2/DQ8; SYBR Green; dried blood spots; duplex reactions; qPCR; type 1 diabetes