基于PCR-SSP技术的RhCE血型基因型检测方法  

Detection of blood RhCE genotype based on PCR-SSP technique

作　　者：陈鲁燕 吕定丰 刘丽平 陈慧敏 陶栓 方野微 徐瑶 张鹤 应琪明 梁伟 CHEN Luyan;LYU Dingfeng;LIU Liping;CHEN Huimin;TAO Shuan;FANG Yewei;XU Yao;ZHANG He;YING Qiming;LIANG Wei(Department of Blood Transfusion,the First Affiliated Hospital of Ningbo University,Ningbo 315000,China;不详)

机构地区：[1]宁波大学附属第一医院输血科,315000 [2]宁波大学附属第一医院检验科,315000 [3]徐州医科大学附属宿迁医院检验科 [4]江苏大学医学院 [5]宁波大学医学部 [6]蚌埠医科大学检验医学院

出　　处：《浙江医学》2024年第19期2021-2026,共6页Zhejiang Medical Journal

基　　金：中国人口福利基金会医学创新课题(SLB-6-20230912-351);宁波市科技局重点项目(2023J020);浙江省医药卫生科技计划项目(2024KY1495)。

摘　　要：目的 基于序列特异性引物PCR(PCR-SSP)技术建立一种用于检测临床标本红细胞RhCE血型基因型的快速定型方法。方法 将2023年9至12月宁波大学附属第一医院收检的152例乙二胺四乙酸抗凝血样分别采用试管法、间接抗人球法和微柱凝胶卡法检测RhD血型和RhCE血型。采用硅基质柱法提取样本DNA。采用Sanger测序法选择RhCE基因标准品序列。采用PCR-SSP法确定检测RhC和Rhc、RhE和Rhe两对等位基因的4对序列特异性引物。采用TA克隆技术得到重组质粒并评估序列特异性引物的灵敏度和抗干扰性能。采用PCR-SSP法检测样本,并与血清学结果进行一致性评价。结果 用表型分别为CCee、CcEe、ccEE的3个标本成功确定不同RhCE基因型的标准品序列。4对序列特异性引物能有效区分RhC和Rhc、RhE和Rhe两对等位基因。序列特异性引物在对应等位基因1:128干扰下仍能检测出且灵敏度为10~4~10~5拷贝数/μL。临床样本RhE、Rhe和Rhc基因型检测结果与表型一致性为100.00%,但RhC基因型与表型一致性只有92.76%,其中RhD阳性背景人群中的一致率为98.39%,RhD阴性背景人群中一致率为88.89%。结论 PCR-SSP技术在红细胞RhCE血型基因型快速鉴定方面具有可行性,检测RhE、Rhe和Rhc基因型与表型具有较高的一致性,对RhC基因型的检测,需区分不同RhD血型背景,建议使用两种及以上的方法进行综合判断。Objective To establish a rapid genotyping method for RhCE blood group based on PCR-sequence specific primer(PCR-SSP)technique.Methods A total of 152 EDTA-anticoagulant blood samples were collected from the First Affiliated Hospital of Ningbo University from September to December 2023.RhD and RhCE blood types were detected by tube method,indirect anti-human sphere method and microcolumn gel card method,respectively.DNA was extracted from the samples using silico matrix column method.Sanger sequencing was used to select the RhCE gene standard sequence.The PCR-SSP technique was used to determine four sequence-specific primers for the detection two pairs of allele of RhC and Rhc,RhE and Rhe genes.TA cloning technique was used to obtain recombinant plasmids,and the sensitivity and anti-interference performance of sequence-specific primers were evaluated.The genes in all blood sampes were detected wity PCR-SSP technique,and the consistency between PCR-SSP and serological results was evaluated.Results The standard sequences of different RhCE genotypes were successfully determined by using three specimens with phenotypes of CCee,CcEe and ccEE,respectively.Four sequence-specific primer pairs were effective in distinguishing among RhC and Rhc,RhE and Rhe alleles.Sequence-specific primers were detected with the interference of the corresponding allele 1:128. The sensitivity ranged from 104 to 105 copies/μL. The consistence rate between RhE, Rhe and Rhc genotype detection and phenotype results was 100.00% in clinical samples, while the cosistence rate for RhC genotype was 92.76%, that was 98.39% and 88.89% in RhD positive and negative background population, respectively. Conclusion PCR-SSP technique is feasible in rapid identification of RhCE blood group genotype in clinical samples. The results of RhE, Rhe and Rhc genotypes are all consistent with the phenotype results. However, RhD blood group background will affect RhC genotyping results, so two or more methods are needed for the acurate detection.

关 键 词：RhCE血型 序列特异性引物PCR 基因分型 单核苷酸多态性 

分 类 号：R457.11[医药卫生—治疗学]