使用Leader引物和FR1引物检测IGHV突变的结果比较  

Comparison of the Results Using Leader and FR1 Primers for IGHV Mutation

作　　者：周茉 陈艳[2] 姚利[2] 王谦[2] 曹杨琳 ZHOU Mo;CHEN Yan;YAO Li;WANG Qian;CAO Yang-lin(Yancheng School of Clinical Medicine of Nanjing Medical University,Yancheng Third People's Hospital,Yancheng,Jiangsu,224001,China;National Clinical Research Center for Hematologic Diseases,Key Laboratory of Thrombosis and Hemostasis of Ministry of Health,Jiangsu Institute of Hematology,First Affiliated Hospital of Soochow University,Suzhou,Jiangsu,215006,China)

机构地区：[1]南京医科大学盐城临床医学院,盐城市第三人民医院血液科,江苏盐城224001 [2]苏州大学附属第一医院,江苏省血液研究所,国家血液系统疾病临床医学研究中心,国家卫生健康委员会血栓与止血重点实验室,江苏苏州215006

出　　处：《中国血液流变学杂志》2022年第4期516-520,608,共6页Chinese Journal of Hemorheology

基　　金：国家自然科学基金资助项目(81970142);苏州市科技发展计划项目(SLJ2021004)。

摘　　要：目的比较Leader引物和FR1引物检测免疫球蛋白重链基因可变区(IGHV)体细胞高频突变的差别,优化IGHV突变检测流程。方法收集2021年9月—2022年8月苏州大学附属第一医院血液科就诊的187例患者的IGHV突变标本,分别使用Leader引物和FR1引物进行PCR扩增、基因测序(单克隆峰使用Sanger测序,双克隆峰使用TA克隆测序),将序列与免疫球蛋白数据库中已知的胚系基因标准序列进行比较,以确定IGHV突变状态,比较不同引物扩增后结果的一致性。结果送检的187例标本中单克隆166例,双克隆21例。统计结果显示,使用Leader引物的阳性率(89.76%)高于使用FR1引物的阳性率(84.94%),两者差异无统计学意义(P>0.05)。两种扩增后的PCR产物中位长度分别为295 bp和252 bp,使用Leader引物扩增的V区完整性(100%)高于使用FR1引物扩增的V区完整性(85.62%),两种引物的使用差异有统计学意义(P<0.0001)。准确性方面,使用Leader引物和FR1引物对结果判读整体上差异无统计学意义(P=0.106),而当同源性百分比处于判读的阈值以上范围≥98%和临界范围97%~99%时,两者差异有统计学意义(P=0.003、P=0.008),表明使用Leader引物的结果准确性要高于使用FR1引物的结果准确性。结论IGHV突变检测时,建议优先使用Leader引物,这样的检测体系准确、快速,能显著提高报告单时效。Objective To compare the difference between the Leader and FR1 primers and optimize the detection process for detecting the mutational status of immunoglobulin heavy chain variable region(IGHV).Methods The IGHV mutation specimens from 187 patients treated in the department of hematology of the First Affiliated Hospital of Soochow University from September 2021 to August 2022 were collected,PCR amplification and sequencing(Sanger sequencing for monoclonal peaks and TA clone sequencing for biclonal peaks)by using Leader primers and FR1 primers.The PCR sequences were compared with the germline sequences in the immunoglobulin database to determine the IGHV mutation status and compare the results'consistency after amplification with different primers.Results There were 166 monoclonal and 21 biclonal specimens among the 187 patients.The statistical analysis showed that the positive rate using Leader primers(89.76%)was higher than that using FR1 primers(84.94%),and there was no statistical difference between them(P>0.05).The median length of amplified PCR products was 295 bp and 252 bp.The V-coverage identity using Leader primers was higher(100%)than that using FR1 primers(85.62%).There was a very significant difference(P<0.0001)in both sequence length and V-coverage identity of the products.Regarding accuracy,the interpretation of the results did not differ in the two primers(P=0.106).At the same time,there were significant differences when the range of cut-off was≥98%,and critical was 97%-99%(P=0.003,P=0.008),indicating the accuracy of the results with the Leader primers was higher than that with FR1 primers.Conclusion Preferentially using Leader primers for IGHV mutation detection is recommend,which is an accurate and rapid experimental method and can effectively improve the timeliness of reports.

关 键 词：IGHV突变 Leader引物 FR1引物 

分 类 号：R734.2[医药卫生—肿瘤]