18-OR: QUANTITATIVE PCR FOR KILLER-CELL IMMUNOGLOBULIN-LIKE RECEPTORS (KIRS) RESEARCH GENOTYPING

Aim Natural Killer cells cause elimination of malignant and virus-infected cells by their cell surface receptors namely Killer-cell Immunoglobulin-like Receptors (KIR). Current KIR genotyping methods use sequence-based typing or sequence-specific primer or oligonucleotide probe (SSP/SSO) based PCR followed by agarose gel electrophoresis. Our aim was to evaluate the feasibility of using a quantitative PCR (qPCR) based research assay for KIR genotyping with minimal DNA input and reduced turn around time with potential for a multiplex KIR genotyping assay in a single tube. Methods A panel of 24 DNA containing common KIR genotypes was obtained from UCLA. 85 ng total DNA for 16 KIR reactions was used for the qPCR research assays compared to 2-3 μg DNA in a SSP-PCR commercial kit. DNA genotypes were confirmed with a SSP-PCR commercial kit and results were compared using the developed qPCR research assay. 32 primers and 16 FAM labeled probes were designed for this assay. The same primer set was also validated using a SYBR Green qPCR assay. A qPCR internal positive control accounts for true absence of KIR alleles. Results KIR research qPCR and SYBR Green qPCR assay genotyping results were 100% concordant with the commercial SSP-PCR kit. Our developed assay could identify the presence or absence of each KIR allele with a 35× reduction in DNA input with no post-PCR analyses or additional specialized equipment. Conclusions We have developed, optimized and validated a qPCR research assay for KIR genotyping that is robust and flexible for both small and large scale genetic analysis studies with potential for high throughput protocols. With more associations of KIR and disease outcomes in autoimmunity, infectious diseases, pregnancy-related complications and in transplantation, a more efficient and sensitive qPCR assay with faster detection might aid earlier therapy intervention making genotyping KIRs using qPCR based assays ideal in clinical and in basic research settings. Wadia: Quest Diagnostics: Employee; Stockholder. Bost: Quest Diagnostics: Employee.