State Key Laboratory of Genetic Engineering, School of Life Science, Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China.
Biomed Res Int. 2018 Mar 7;2018:6828306. doi: 10.1155/2018/6828306. eCollection 2018.
Hearing loss is an etiologically heterogeneous trait with a high incidence in China. Though conventional newborn hearing screening program has been widely adopted, gene detection can significantly improve the means of early discovering genetic risk factors. Thus, simple and efficient methods with higher sensitivity and lower cost for detecting hotspot mutations of hearing loss are urgently requested. Here we established a mutation detection system based on multiple fluorescent probe technique, which can detect and genotype nine hotspot mutations of four prominent hearing loss-related genes in two reactions on a four-channel real-time PCR instrument, including (rs750188782, rs80338943, rs1110333204, and rs80338939), (rs74315319), (rs111033313 and rs121908362), and (rs267606617 and rs267606619). This system is with high sensitivity that enables detecting as low as 10 DNA copies samples per reaction. A comparison study in 268 clinical samples showed that the detection system had 100% concordance to Sanger sequencing. Besides, blood and saliva samples can be directly detected without DNA extraction process, which greatly simplifies the manipulation. The new system with high sensitivity, accuracy, and specimen type compatibility can be expectedly a reliable tool in clinical application.
听力损失是一种病因异质性的特征，在中国发病率很高。尽管传统的新生儿听力筛查计划已经广泛采用，但基因检测可以显著提高发现遗传风险因素的早期手段。因此，迫切需要简单、高效、灵敏度更高、成本更低的方法来检测听力损失热点突变。在这里，我们建立了一种基于多重荧光探针技术的突变检测系统，该系统可以在四通道实时 PCR 仪器上的两个反应中检测和分型四个主要听力损失相关基因的九个热点突变，包括 (rs750188782、rs80338943、rs1110333204 和 rs80338939)、 (rs74315319)、 (rs111033313 和 rs121908362) 和 (rs267606617 和 rs267606619)。该系统具有很高的灵敏度，每个反应可以检测低至 10 个 DNA 拷贝的样本。在 268 个临床样本的比较研究中，该检测系统与 Sanger 测序具有 100%的一致性。此外，无需进行 DNA 提取过程，即可直接检测血液和唾液样本，大大简化了操作。该系统具有高灵敏度、准确性和标本类型兼容性，有望成为临床应用中一种可靠的工具。
