Establishment of Q-PCR method for qualitative testing of the presence or absence of KIR genes

Abstract

Objective To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR). Methods Based on the polymorphism of high-resolution level KIR alleles in Chinese population and the IPD-KIR database, KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR. Meanwhile, one negative control and one positive control specific amplifying human growth hormone (HGH) gene fragment were set to monitor the false positive and false negative results in PCR amplification, respectively. A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by authors. Results The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results, but two samples showed inconsistent results. One sample was negative for 2DS5 by Q-PCR but positive by PCR-SSP, another sample was positive for 2DS1 by Q-PCR but negative by PCR-SSP. The two doubtful samples were genotyped by sequencing-based typing (PCR-SBT) for 2DS5 and 2DS1, respectively. PCR-SBT results confirmed that the results of Q-PCR test was correct. Conclusion The KIR Q-PCR method established in this paper can provide accurate and reliable results for testing the presence or absence of KIR genes.

Keywords

• killer-cell immunoglobulin-like receptor (kir)
• presence or absence of kir genes
• quantitative real time-pcr (q-pcr)
• sequence specific primers-pcr (pcr-ssp)
• sequencing-based typing (pcr-sbt)