α-地中海贫血和β-地中海贫血基因突变异尾双链荧光探针杂交法的建立及应用评价  

Establishment and evaluation ofα-thalassemia andβ-thalassemia mutation determinations using hetero-tail dual-labeled fluorescent probe hydridization

作　　者：李仁强 罗俊峰 陈云弟 LI Renqiang;LUO Junfeng;CHEN Yundi(Department of Research and Development,Shanghai NuProbe Gene Technology Limited Company,Shanghai 200433,China)

机构地区：[1]上海阅尔基因技术有限公司技术研发部,上海200433

出　　处：《检验医学》2022年第10期955-962,共8页Laboratory Medicine

摘　　要：目的 建立可同时检测3种α-地中海贫血(简称地贫)非缺失突变(αWS、αQS、αCS)和8种β-地贫基因点突变[CD26(G>A)、CD41/42(-TCTT)、nt29(A>G)、IVS-Ⅱ-654(C>T)、nt28(A>G)、CD71/72(+A)、CD17(A>T)、CD27/28(+C)]的异尾双链荧光探针杂交法,并评价其临床应用价值。方法设计目标区域的聚合酶链反应(PCR)引物和异尾双链荧光探针,对PCR反应体系和反应条件进行优化。采用地贫核酸检测国家参考品评估异尾双链荧光探针杂交法的检测性能(准确性、特异性、灵敏度)。采用异尾双链荧光探针杂交法和PCR-反向点杂交法同时检测200份已知地贫基因型的临床样本,评估2种方法检测结果的符合率。结果 对于检测范围内的11种点突变样本,异尾双链荧光探针杂交法检测结果与国家参考品说明书标示的突变类型的符合率为100%;检测范围外的其他点突变或缺失突变类型均未检出。采用异尾双链荧光探针杂交法检测1.0、2.5、5.0、10.0 ng/μL的α-地贫Hb CS点突变参考品和β-地贫CD41/42、IVS-Ⅱ-654点突变参考品,结果均与国家参考品说明书中标示的突变类型一致。异尾双链荧光探针杂交法与PCR-反向点杂交法检测结果的符合率为100%。结论 异尾双链荧光探针杂交法仅需1步加样,闭管检测,操作简单,无污染,来检测时间较短,具有一定的临床应用潜力。Objective To establish and evaluate a method for determining 3α-thalassemia non-deletional mutations(αWS,αQS,αCS)and 8β-thalassemia point mutations[CD26(G>A),CD41/42(-TCTT),nt29(A>G),IVS-Ⅱ-654(C>T),nt28(A>G),CD71/72(+A),CD17(A>T),CD27/28(+C)]simultaneously using hetero-tail dual-labeled fluorescent probe hydridization.Methods Polymerase chain reaction(PCR)primers and hetero-tail dual-labeled fluorescent probes targeting 11 most commonα-thalassemia non-deletional andβ-thalassemia point mutations were designed.An optimized multiplex asymmetric PCR and hetero-tail dual-labeled fluorescent probe hydridization were established.Performance specifications(accuracy,specificity and sensitivity)were evaluated by the national reference materials for thalassemia.Totally,200 clinical samples were determined by PCR-reverse dot blot kit and hetero-tail dual-labeled fluorescent probe hydridization concurrently to assess the consistency.Results For 11 mutations within the determination range,hetero-tail dual-labeled fluorescent probe hydridization showed 100%consistency with the national reference materials for thalassemia,and none of the mutations outside the determination range were determined.For different concentrations of Hb CS,CD41/42 and IVS-Ⅱ-654 reference materials ranging from 1.0,2.5,5.0 to 10.0 ng/μL,the results were consistent.The consistency rate between PCR-reverse dot blot kit and hetero-tail dual-labeled fluorescent probe hydridization was 100%.Conclusions Hetero-tail dual-labeled fluorescent probe hydridization enables fast and accurate determination of 11α-thalassemia non-deletional andβ-thalassemia point mutations simultaneously by one-step real-time PCR,which reduces turn-around time and contamination risk and shows potential for future clinical application.

关 键 词：异尾双链荧光探针 多重不对称聚合酶链反应 非缺失突变 点突变 地中海贫血 

分 类 号：R446.5[医药卫生—诊断学]