1031-LBP: Evaluation of uracil dna glycosylase (UDG) with the LinkSeq™ real-time pcr hla typing method

Abstract

Aim Post-PCR amplification products can pose a problem in clinical laboratories. Great care is needed to ensure post and pre-PCR areas minimize the risk of unwanted amplicons affecting results. Linkage Biosciences designed the LinkS e ¯ q™ real-time PCR HLA test to include deoxyuridine triphosphate (dUTP) rather than the standard deoxythmidine triphosphate (dTTP) in the PCR reaction as a preventive measure so that Uracil DNA Glycosylase enzyme (UDG) can be used as an optional additive to eliminate unwanted uracil-containing amplicons during PCR set-up. The aim of this study was to evaluate the effectiveness of adding UDG to the LinkS e ¯ q™ test in a laboratory setting with False Positive and False Negative issues. Methods To validate the usefulness of UDG reagent (provided by Linkage Biosciences) we selected 10 clinical DNA samples that contained aberrant primer dimer peaks which may have contributed to False Positive and False Negative allele amplification. All samples were typed with the standard LinkS e ¯ q™ protocol and with the UDG protocol. The 2 real-time kits that were used were the LinkS e ¯ q™ 384 well tray, Cat #1575 and the 192 well tray, Cat#1554 with SYBR Green dye for melting curve analysis. All samples were amplified using the Roche LightCycler® 480. Results All 10 samples showed improved results in the reduction of False Positives and removal of dimeric peaks using the UDG reagent. The data shows a total reduction of 93.6% of the False Positives, 66.6% reduction in False Negatives, and 86.7% reduction of software forced calls. The samples demonstrated concordance with PCR-SSP (Invitrogen), or UNET reported results. Conclusions In summary, the LinkS e ¯ q™ real-time PCR assay is a fast and reliable HLA typing method. The UDG reagent works well when necessary to remove suspect peaks which may interfere with SureTyper Software data analysis.