Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods.
Quality control (QC) in laboratories performing molecular histocompatibility class II typing often includes a polymerase chain reaction (PCR) approach for monitoring DNA contamination. An oligonucleotide primer set was designed, (RBQBf/RBQBr), which is specific for nonpolymorphic regions of the DR-B, DQ-B, and DP-B consensus sequences with an expected PCR product size of 81 bp. RBQBf/RBQBr detected genomic DNA from reference cell lines LWAGS and BM21 (50 to 100 picograms) as well as DR-B, DP-B, and DQ-B amplicon (1 copy). Additionally, RBQBf/RBQBr detected SSP products from routine DR-B and DQ-B typings. Validation studies employing controlled DNA contamination of laboratory surfaces revealed that increasing amounts of wipe test samples (5% to 20% v/v) were inhibitory to the wipe test PCR, whereas lower amounts (1% to 2%), or alternatively, a diluted wipe test sample, increased the sensitivity of the test and optimized the results. Collectively, this study describes a primer set, RBQBf/RBQBr, which detects both genomic DNA and DR-B, DQ-B, or DP-B amplicon and furthermore illustrates the necessity of routine testing for potential inhibitory factors that may be introduced into the wipe test PCR.
McCormack
J M
JM
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Sherman
M L
ML
Maurer
D H
DH
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000592:standards; D005188:False Negative Reactions; D005189:False Positive Reactions; D006681:HLA-D Antigens / Q000235:genetics*; D006650:Histocompatibility Testing / Q000592:standards*; D006801:Humans; D007753:Laboratories; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000592:standards*; D011786:Quality Control; D015203:Reproducibility of Results
D017931:DNA Primers; D006681:HLA-D Antigens; D004247:DNA