微流控芯片在遗传性耳聋基因热点突变检测分析中的应用  

Validation of microfluidic chip for detection of mutation hotspots associated with hereditary hearing loss

作　　者：张冠斌 邹凌 王国建[3] 杨佳文 梁冬 俞皓 邓霖 尹维[5] 方雪 Guanbin Zhang;Ling Zou;Guojian Wang;Jiawen Yang;Dong Liang;Hao Yu;Lin Deng;Wei Yin;Xue Fang(Chengdu Capitalbio MedLab,Chengdu 611130,China;Chengdu Women′s and Children′s Central Hospital,School of Medicine,University of Electronic Science and Technology of China,Chengdu 611731,China;Department of Otolaryngology Head and Neck Surgery,Chinese People′s Liberation Army(PLA)General Hospital,Beijing 100853,China;Chengdu Capital bioJinxin Bio-Technology,Chengdu 611130,China;Mianyang People′s Hospital,Mianyang 621000,China)

机构地区：[1]成都博奥独立医学实验室,成都611130 [2]电子科技大学医学院附属妇女儿童医院·成都市妇女儿童中心医院,成都611731 [3]解放军总医院耳鼻咽喉头颈外科,北京100853 [4]成都博奥晶芯生物科技有限公司,成都611130 [5]绵阳市人民医院,绵阳621000

出　　处：《中华检验医学杂志》2022年第6期616-622,共7页Chinese Journal of Laboratory Medicine

摘　　要：目的探索将微流控芯片用于遗传性耳聋基因热点突变的分型检测。方法将专门设计加工的微流控芯片与KASP扩增整合,借助激光共聚焦荧光扫描仪实现遗传性耳聋常见4个相关基因的23个热点突变分型检测,采集276例(听障组:成都市2019年出生并诊断为听力损失的131例新生儿;对照组:成都市2020年出生且听力筛查结果为双耳通过的145例新生儿)新生儿的足跟血斑样品验证其结果准确性和临床应用可行性。结果微流控芯片通过聚类分析对全部质控品均实现准确分型,其最低检测限可达1 mg/L。276例新生儿血斑样品的微流控芯片分型结果全部通过复核确认。听障组131例新生儿,检出66例阳性携带者,阳性携带率50.4%;对照组145例新生儿,检出40例阳性携带者,阳性携带率27.6%。其中携带率最高的突变热点为GJB2基因c.109G>A,在听障组和对照组中阳性携带率分别为46.6%(61/131)和23.4%(34/145)。结论微流控芯片可用于遗传性耳聋相关热点突变的分型检测,具有准确性高、防污染能力强、操作简单、用时短等优势,能更好地满足新生儿耳聋基因筛查等遗传性耳聋基因检测需求。Objective To explore the application of microfluidic chip in detection of hereditary deafness-associated hotspot mutations.Methods The dedicatedly designed and fabricated microfluidic chip was integrated with kompetitive allele-specific polymerase chain reaction amplification system,scanned via laser-excited confocal fluorescence scanner,and finally analyzed programmatically to acquire the typing results of the 23 mutation sites of the four common genes associated with hereditary hearing loss.Dried blood spots were collected from 276 neonates containing the 131 cases with hearing loss who were born in 2019 in Chengdu(deafness group)and the 145 cases with normal hearing who were born in 2020 in Chengdu(control group),and analyzed by the microfluidic chip to evaluate its clinical performance.Results By cluster analysis,the microfluidic chip correctly analyzed the 23 positive reference samples and acquired the same typing results as their actual results,with a limit of detection of 1 mg/L.For the 276 newborn blood spots,the detection results of the microfluidic chips were confirmed to be correct by the contrasting methods.Among Deafness Group,66(50.4%)tested positive for the selected 23 mutation hotspots;among Control Group,40(27.6%)were positive.Among these mutations,c.109G>A of the GJB2 gene was the most prevalent one,whose carrier rate in deafness group and control group were 46.6%(61/131)and 23.4%(34/145),respectively.Conclusions The micro-fluidic chip system was succeeded in fulfilling the hereditary deafness-related mutation detection,and offered many advantages including high specificity,avoiding the amplicon carryover contamination,simplifying the entire experimental operation process and short detection time,so as to better meet the detection requirement of genetic testing for deafness in newborn screening and other fields.

关 键 词：微流体分析技术 听觉丧失 突变分析 新生儿筛查 

分 类 号：R764.43[医药卫生—耳鼻咽喉科] R440[医药卫生—临床医学]