Establishment of a PCR-SSP method for the simultaneous amplification and identification of the presence or absence ofKIR genes
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目的 探讨建立同步扩增中国人群全部KIR基因和鉴定KIR基因有无的序列特异性引物-聚合酶链反应(PCR-SSP)方法。 方法 选取2015年1月至2015年11月在深圳市血液中心无偿献血的132份健康受试者的血样为研究对象。基于中国人群精细水平KIR等位基因的多态性和单核苷酸多态性(SNP)信息，参考IPD-KIR数据库，设计KIR基因特异性引物用于扩增16种KIR基因及2DS4-Normal、2DS4-Deleted 2种亚型；并采用KIR基因型已知样本，对每对PCR引物的特异性进行验证。在KIR基因的PCR扩增反应中，复合扩增人体生长激素(HGH)基因片段作为内对照，以防止PCR扩增出现假阴性结果。随机选取132份KIR基因型已知的健康受试者DNA样本进行盲检，验证该方法的可靠性。 结果 本研究设计和筛选出的KIR特异性引物，可鉴定相应的KIR基因，而且内对照条带和特异性扩增条带均清晰、明亮；采用本研究建立的鉴定KIR基因有无的PCR-SSP方法检测纳入研究的132份样本的盲检结果，与已知KIR基因型完全一致。 结论 本实验建立的KIR PCR-SSP方法，鉴定中国人群KIR基因的有无可获得准确、可靠的结果。 Objective To develop a polymerase chain reaction-sequence specific primer (PCR-SSP) method for simultaneous amplification and identification of the KIR genes among Chinese population. Methods Peripheral blood samples from 132 healthy donors who had given blood at Shenzhen Blood Center from January 2015 to November 2015 were selected as the study subjects. Based on the polymorphism and single nucleotide polymorphism (SNP) information of high-resolution KIR alleles in the Chinese population and the IPD-KIR database, specific primers were designed to amplify all the 16 KIR genes and the 2DS4-Normal and 2DS4-Deleted subtypes. The specificity of each pair of PCR primers was verified by using samples with known KIR genotypes. During PCR amplification of the KIR gene, co-amplification the fragment of human growth hormone (HGH) gene by multiplex PCR was used as the internal control to prevent false negative results. A total of 132 samples with known KIR genotypes were randomly selected for blind inspection to verify the reliability of the developed method. Results The designed primers can specifically amplify the corresponding KIR genes, with clear and bright bands for the internal control and KIR genes. The results of detection are fully consistent with the known results. Conclusion The KIR PCR-SSP method established in this study can yield accurate results for the identification of the presence or absence of KIR genes.
Objective To develop a polymerase chain reaction-sequence specific primer (PCR-SSP) method for simultaneous amplification and identification of the KIR genes among Chinese population. Methods Peripheral blood samples from 132 healthy donors who had given blood at Shenzhen Blood Center from January 2015 to November 2015 were selected as the study subjects. Based on the polymorphism and single nucleotide polymorphism (SNP) information of high-resolution KIR alleles in the Chinese population and the IPD-KIR database, specific primers were designed to amplify all the 16 KIR genes and the 2DS4-Normal and 2DS4-Deleted subtypes. The specificity of each pair of PCR primers was verified by using samples with known KIR genotypes. During PCR amplification of the KIR gene, co-amplification the fragment of human growth hormone (HGH) gene by multiplex PCR was used as the internal control to prevent false negative results. A total of 132 samples with known KIR genotypes were randomly selected for blind inspection to verify the reliability of the developed method. Results The designed primers can specifically amplify the corresponding KIR genes, with clear and bright bands for the internal control and KIR genes. The results of detection are fully consistent with the known results. Conclusion The KIR PCR-SSP method established in this study can yield accurate results for the identification of the presence or absence of KIR genes.
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