title: Genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples.
abstract: Expansion of molecular diagnostics more widely into clinical routines requires simplified methods allowing automation. We developed a homogeneous, multilabel polymerase chain reaction (PCR) method based on time-resolved fluorometry, and studied the use of dried disk samples in PCR. Celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 genotyping was used to verify the method with blood and saliva samples dried on S&S 903 and IsoCode sample collection papers. Three sample preparation procedures, including manufacturer's manual elution, an automated elution, and direct use of disk samples, were compared using dried disk samples. The three procedures gave successful amplification and correct genotyping results. Owing to the simplicity of the direct use of disk samples in PCR, this method was chosen for the subsequent homogeneous analysis of blood (n=194) and saliva (n=30) disk samples on S&S 903 paper. The results revealed that, in addition to DNA samples (n=29), both blood and saliva disk samples were successfully amplified and genotyped using the homogeneous PCR assays for HLA-DQA1 and HLA-DQB1. The homogeneous PCR assays developed provide a useful tool to genotype celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 alleles. Furthermore, the method provides a direct way to perform a closed-tube PCR analysis of dried blood and saliva disk samples enabling simple automation.
authors.affiliation: PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland.
authors.forename: Pia
authors.initials: P
authors.lastname: Ollikka
authors.forename: Hanna-Mari
authors.initials: HM
authors.lastname: Raussi
authors.forename: Ville
authors.initials: V
authors.lastname: Laitala
authors.forename: Lassi
authors.initials: L
authors.lastname: Jaakkola
authors.forename: Jari
authors.initials: J
authors.lastname: Hovinen
authors.forename: Ilkka
authors.initials: I
authors.lastname: Hemmilä
authors.forename: Alice
authors.initials: A
authors.lastname: Ylikoski
mesh_terms: D001769:Blood; D002446:Celiac Disease / Q000235:genetics*; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics*; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; D006239:Haplotypes; D006801:Humans; D008722:Methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012107:Research Design; D012463:Saliva / Q000737:chemistry
chemical_list: D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; C069050:HLA-DQA1 antigen; C069051:HLA-DQB1 antigen