应用Q-PCR定性检测KIR基因有无方法的建立  

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

作　　者：李宇楠 甄建新[2] 梁爽 喻琼[1] 邓志辉[1] LI Yunan;ZHEN Jianxin;LIANG Shuang;YU Qiong;DENG Zhihui(Institute of Blood Transfusion,Shenzhen Blood Center,Shenzhen 518040,China;Central Laboratory,Shenzhen Baoan Women′s and Children′s Hospital)

机构地区：[1]深圳市血液中心输血医学研究所,广东深圳518040 [2]深圳市宝安区妇幼保健院中心实验室

出　　处：《中国输血杂志》2024年第6期660-665,共6页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金青年科学基金项目(82203291);广东省基础与应用基础研究基金委员会项目(2022A1515011045);深圳市科技创新委员会基础研究项目(JCYJ20190806152001762);深圳市医学重点学科(SZXK070)。

摘　　要：目的建立定性检测KIR基因有无的Q-PCR方法。方法根据高分辨水平中国人群KIR等位基因的多态性,并参考国际IPD-KIR数据库,针对16种KIR基因及2DS4-Normal、2DS4-Deleted两种亚型,设计KIR基因特异性引物用于Q-PCR扩增反应;同时设置一孔阴性对照、一孔阳性对照(特异性扩增人体生长激素HGH基因片段),以监控假阳性、假阴性的结果。为验证Q-PCR方法的可靠性,随机选择302份已采用KIR PCR-SSP商品化试剂盒检测的标本,采用Q-PCR方法盲检和对比。结果300人份的Q-PCR检测结果与已知的PCR-SSP检测结果相符,有2份标本结果不一致,其中1例标本的2DS5基因Q-PCR检测结果为阴性,而PCR-SSP检测结果为阳性;另一例标本2DS1基因Q-PCR检测结果为阳性,而PCR-SSP检测结果为阴性。对2份标本分别进行2DS5、2DS1基因测序分型,证实Q-PCR定性检测结果正确。结论本文建立的KIR Q-PCR方法结果准确、可靠,可用于KIR基因有无的定性检测。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.

关 键 词：杀伤细胞免疫球蛋白样受体(KIR) KIR基因有无 实时荧光定量-PCR 序列特异性引物-PCR 测序分型 

分 类 号：R457.1[医药卫生—治疗学] Q811.4[医药卫生—临床医学]