PCR-高分辨熔解曲线法在Kidd血型基因分型中的应用  

Application of PCR-high resolution melting curve analysis in Kidd blood group genotyping

作　　者：章昊[1] 林坤达 梁爽 苏宇清[1] 洪文旭[1] Zhang Hao;Lin Qunda;Liang Shuang;Su Yuqing;Hong Wenxu(Shenzhen Blood Center,Shenzhen 518035,Guangdong Province,China;School of Life and Marine Sciences,Shenzhen University,Shenzhen 518060,Guangdong Province,China)

机构地区：[1]深圳市血液中心,518035 [2]深圳大学生命与海洋科学学院,518060

出　　处：《国际输血及血液学杂志》2021年第1期78-83,共6页International Journal of Blood Transfusion and Hematology

基　　金：深圳市科技计划项目(JCYJ20180302153621300);深圳市卫健委医学重点学科项目(SZXK070)。

摘　　要：目的探讨PCR-高分辨熔解曲线(HRM)法在Kidd血型基因分型中的应用。方法采用简单随机抽样法,选择2019年10月至11月,于深圳市血液中心参加无偿献血的256例献血者为研究对象。本组献血者年龄为18~60岁;男性献血者为142例,女性为114例。采用血型血清学试验方法对本组献血者全血标本的红细胞抗原表型进行检测。采用PCR-SSP和PCR-HRM法对本组献血者的DNA标本进行Kidd血型基因分型。对于基因分型与血型血清学检测结果不一致的标本,则通过JK基因第9外显子直接测序法进行确认。本研究遵循的程序符合2013年修订版《世界医学协会赫尔辛基宣言》要求,并且于献血前与所有献血者签署《献血者知情同意书》。结果①本组256例无偿献血者全血标本的血型血清学检测结果显示,Jk(a-b+)、Jk(a+b+)和Jk(a+b-)表型献血者分别为86、109和61例。②本组256例献血者基因组DNA标本的PCR-SSP和PCR-HRM法的Kidd血型基因分型结果一致,JKB/JKB、JKA/JKB和JKA/JKA基因型献血者分别为82、115和59例。③22例(8.6%,22/256)献血者的血型血清学表型与基因分型结果不一致。其基因组DNA标本经JK基因第9外显子直接测序结果显示,JK基因第9外显子存在c.838 G>A突变,与JKA/JKB基因分型结果相符。结论本研究建立的PCR-HRM法可准确进行Kidd血型基因分型,该方法可以有效地辅助Kidd血型精准鉴定。在进行Kidd血型鉴定的实际工作中,可联合血型血清学和PCR-HRM法,以提高Kidd血型鉴定准确度。Objective To explore the application of PCR-high resolution melting curve(HRM)analysis in Kidd blood group genotyping.Methods From October to November 2019,a total of 256 donors who participated in voluntary blood donation in Shenzhen Blood Center were selected as subjects by simple random sampling method.The age of these voluntary blood donors was 18 to 60 years old.There were 142 male donors,and 114 females.Serological test was used to detect the erythrocyte antigen phenotype of whole blood specimens from all 256 voluntary blood donors.PCR-sequence specific primer(SSP)and PCR-HRM analysis methods were used to detect the Kidd blood group genotyping of DNA specimens from all blood donors,respectively.Direct sequencing of JK gene exon 9 was used to confirm the genotype of the specimens,whose genotyping and serological phenotype results were inconsistent.The procedure followed in this study were in accordance with the requirements of the World Medical Association Declaration of Helsinki revised in 2013.And the Informed Consent of Blood Donors were signed with all blood donors before donation.Results①Among all 256 whole blood specimens,there were 86 cases with Jk(a-b+),109 cases with Jk(a+b+),and 61 cases with Jk(a+b-)phenotype.②The results of 256 DNA specimens between PCR-SSP and PCR-HRM analysis were consistent.And there were 82 blood donors with JKB/JKB genotype,115 cases with JKA/JKB genotype,59 cases with JKA/JKA genotype.③There were 22 blood donors(8.6%,22/256)whose serological phenotypes were inconsistent with genotyping.Direct sequencing results of JK gene exon 9 from these 22 inconsistent specimens were identified that there was a c.838 G>A mutation in JK gene exon 9,which were consistent with Kidd genotype results of PCR-SSP and PCR-HRM analysis.Conclusions The PCR-HRM analysis method established in this study could accurately perform genotyping for JK gene,and it can effectively assist in precisely identification of Kidd blood group typing.In the actual work of Kidd blood group identification,combin

关 键 词：KIDD血型系统 基因型 基因分型技术 多态性 单核苷酸 高分辨率熔解曲线 基因分型 JK基因 

分 类 号：R457.11[医药卫生—治疗学]