摘要
目的应用实时荧光定量PCR(real-time-qPCR)对全国范围内的耳聋患者进行mtDNA C1494T的筛查,为进一步研究该突变和聋病的临床基因诊断提供帮助。方法收集国内25个不同省市的特教学校和聋哑学校的3 133例双侧重度或极重度非综合征型聋患者,提取外周血DNA,设计TaqMan-MGB的引物和探针进行real-time-qPCR,对所有阳性样本和随机抽取的495例阴性样本进行直接测序验证。结果 3 133例中,发现C1494T突变14例(14/3 133,0.45%),经测序验证全部为C1494T突变,495例阴性样本中均无C1494T突变。结论 Real-time-qPCR是进行mtDNA C1494T突变筛查的有效方法,中国耳聋人群中mtDNA C1494T的突变率较低。
Objective To develop a simple, rapid, and reliable real--time quantitative polymerase chain reaction （real--time qPCR） assay based on TaqMan technology using a new minor groove binding （MGB） probe for directly detecting the mitochondrial DNA （mtDNA） C1494T mutation, and to investigate the carrier frequency in nonsyndromic deaf Chinese subjects. Methods A TaqMan--MGB probe was constructed. Peripheral blood samples were collected from 3 133 nonsyndromic deaf patients and genomic DNA was extracted. A real--time qPCR using MGB probes （wild--type） in a single tube was used to detect the mtDNA C1494T mutation. The results were then compared to the DNA sequence of the PCR products. Results A total of 13 of 3 133 （0. 45%） Chinese nonsyndromie hearing loss patients were C1494T--positive. The results of the TaqMan- MGB probe method were consistent with those of sequencing. Conclusion Real--time qPCR with a TaqMan MGB probe is useful for large--scale screening for screening of the C1494T mutation. The mtDNA C1494T mutation has a low carrier frequency in Chinese nonsyndromic hearing loss patients.
出处
《听力学及言语疾病杂志》
CAS
CSCD
北大核心
2013年第1期23-26,共4页
Journal of Audiology and Speech Pathology
基金
江苏省科教兴卫医学重点人才(RC2011028)
卫生部行业科研专项基金(201202005)
南京市医学科技发展基金重点项目(ZKX09009)
国家自然科学基金(30572015
30728030
31071109)联合资助
