RHCE~* ce(308C>T)突变型等位基因Taqman实时荧光定量PCR检测方法的建立及应用  

Establishment and application of Taqman real-time quantitative PCR method for RHCE~* ce( 308C>T) mutant allele

作　　者：王贞[1] 贾双双[1] 廖志坚[1] 张润青[1] 罗广平[1] 姬艳丽[1] WANG Zhen;JIA Shuangshuang;LIAO Zhijian;ZHANG Runqing;LUO Guangping;JI Yanli(Institute of Clinical Trans-fusion,Guangzhou Blood Center,Guangdong,Guangzhou 510095)

机构地区：[1]广州血液中心临床输血研究所

出　　处：《中国输血杂志》2018年第9期926-929,共4页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金(81500155);广州市医学重点实验室项目

摘　　要：目的建立Rh血型系统RHCE＊ ce（308C〉T）突变型等位基因的Taqman探针实时荧光定量PCR检测方法,筛查携带该等位基因的个体,并对其Rh CE血型抗原进行鉴定分析。方法针对RHCE基因c. 308C〉T突变位点设计特异性引物及Taqman-MGB探针,采用已知携带该突变位点的标本作为阳性对照,建立实时荧光定量PCR方法。应用该方法对800例RhD阴性标本、1 000例RhD阳性标本以及400例D放散型标本进行突变筛查,同时随机抽取200例标本进行RHCE基因外显子2直接测序。对筛查出携带该突变型等位基因的标本进行RHCE基因外显子2测序确证,同时应用多重连接依赖式探针扩增（MLPA）基因检测结合测序方法对其进行Rh血型基因型鉴定,并进行Rh血型血清学检测。此外,采用6种针对不同抗原表位的单克隆抗体对Rhc抗原的表达进行血清学鉴定,并采用4种抗-c进行Rhc抗原的流式细胞检测。结果成功建立RHCE＊ ce（308C〉T）等位基因的Taqman探针实时荧光定量PCR方法。应用该方法从800例RhD阴性标本中筛查出2例携带该突变型等位基因标本,RHCE基因外显子2测序结果证实存在杂合点突变（c. 308C〉T,p. 103Pro〉Leu）,但在RhD阳性及D放散型标本中未检出此突变。此2例标本的Rh抗原表型均为CCdee,基因型均为Cde/c^vde[c^v表示RHCE＊ ce（308C〉T）突变型等位基因]。该2例标本的Rhc抗原血清学和流式细胞检测均为阴性,而Rh C抗原表达正常。结论 Taqman探针实时定量PCR方法进行RHCE＊ ce（308C〉T）等位基因检测快速准确,该等位基因可导致c抗原不表达。Objective To establish a Taqman probe real-time quantitative PCR assay for the RHCE＊ ce（308C〉T） mu- tant allele. Screen the individuals carrying this allele and analyze the RhCE antigen. Methods A specific primer and Taq- man-MGB probe were designed for the c.308C〉T mutation site of RHCE gene. Samples carrying the mutation site were usedas a positive control to establish a real-time PCR method. With this method, 800 RhD negative samples, 1 000 RhD positive samples and 400 Asian type DEL samples were screened for mu- tation. Meanwhile, 200 samples were randomly selected for the direct sequencing of exon 2 of the RHCE gene. The samples car- rying the mutant allele were re-confirmed by the sequencing re- suits. The Rh genotype was identified by Multiplex Ligation-de- pendent Probe Amplification （MLPA） combined with sequen-cing, and serological tests were performed. In addition, the expression of Rhc antigen was serologically identified using six monoclonal antibodies against different epitopes, and four anti-c antibodies were used for flow cytometry of Rhc antigen. Re- suits The Taqman probe real-time quantitative PCR method for RHCE＊ce（308C〉T） allele was successfully established. In this method, 2 samples carrying the mutant allele were identified from 800 RhD-negative samples. The sequencing of exon2 confirmed the presence of heterozygous mutations （c.308C〉T, p.103Pro〉Leu）. However, this mutation was not detected in the RhD positive and DEL samples. The Rh phenotypes of both samples were CCdee, and the genotypes were Cde/cvde （ cv indicates the RHCE＊ce（308C〉T） mutant allele）. The serology and flow cytometry tests of the Rhc antigen in both samples were negative, while RhC antigen expression was normal. Conclusion The Taqman probe real-time quantitative PCR meth- od is rapid and accurate for the detection of RHCE＊ce（308C〉T） alleles, which can cause the non-expression of the c anti- gen.

关 键 词：RHCE基因 Taqman探针实时荧光定量PCR RHCE＊ ce(308C〉T) 

分 类 号：R457.11[医药卫生—治疗学]