荧光PCR熔解曲线法联合Sanger测序技术在新生儿耳聋基因筛查中的应用

潘澍青; 潘婕文; 鲍幼维; 潘小莉; 庄丹燕

doi:10.16766/j.cnki.issn.1674-4152.003965

荧光PCR熔解曲线法联合Sanger测序技术在新生儿耳聋基因筛查中的应用

doi: 10.16766/j.cnki.issn.1674-4152.003965

宁波大学附属妇女儿童医院出生缺陷综合防治中心宁波市胚胎源性疾病防治重点实验室宁波市基因组医学与出生缺陷防治重点实验室，浙江宁波 315012

基金项目:

浙江省医药卫生科技计划项目 2023KY1121

宁波市医学重点扶植学科儿童保健学 2022-F26

详细信息

通讯作者:
庄丹燕，E-mail：nbsclab@126.com

中图分类号: R764.43 R446.7
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出版历程
- 收稿日期: 2024-06-11
- 网络出版日期: 2025-06-30

Application of the real-time fluorescence PCR melting curve method in gene screening for newborn hearing loss

The Central Laboratory of Birth Defects Prevention and Control, Women and Children' s Hospital Affiliated to Ningbo University, Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, Ningbo, Zhejiang 315012, China

摘要

摘要: 目的分析荧光PCR熔解曲线法在新生儿耳聋基因筛查中的准确性，了解本地区常见耳聋基因的变异类型及频率，为今后耳聋基因筛查和耳聋防控提供参考。方法回顾性选取2022年11月—2023年10月于宁波大学附属妇女儿童医院分娩，并进行耳聋基因检测的5 563例新生儿。采用荧光PCR熔解曲线法检测4个常见耳聋基因(GJB2、SLC26A4、线粒体12S rRNA和GJB3)的15个变异位点，再采用Sanger测序验证阳性样本。结果 5 563例新生儿中，检出耳聋基因变异256例，总检出率为4.60%，其中GJB2基因变异156例，检出率为2.80%；SLC26A4基因变异73例，检出率为1.31%；线粒体12S rRNA基因变异10例，检出率为0.18%；GJB3基因变异16例，检出率为0.29%；同时检出1例复合杂合变异，检出率为0.02%。阳性样本经Sanger测序验证，发现9例结果不相符，因此，荧光PCR熔解曲线法的准确率为96.48%。结论本地区新生儿耳聋基因变异以GJB2和SLC26A4基因为主；荧光PCR熔解曲线法联合Sanger测序法可准确检出耳聋基因变异情况，有助于及早发现先天性、药物性和迟发性耳聋，对早期预防和早期治疗耳聋具有重要意义。
- 耳聋基因 /
- 基因变异 /
- Sanger测序 /
- 新生儿
Abstract: Objective To analyze the accuracy of the fluorescence PCR melting curve method in screening for newborn deafness genes, and to understand the variation types and frequencies of common deafness genes in the local population. It provides reference for gene screening and prevention of deafness in the future. Methods A retrospective analysis was conducted on 5 563 newborns and screened for deafness genes at Ningbo Women and Children' s Hospital Affiliated to Ningbo University, from November 2022 to October 2023. Firstly, the fluorescent PCR melting curve method was used to detect 15 mutation sites in four deafness genes (GJB2, SLC26A4, mitochondrial 12S rRNA, and GJB3), followed by Sanger sequencing to verify the positive samples. Results Among the 5 563 newborns, 256 cases of deafness gene mutation were detected, yielding a total detection rate of 4.60%. Mutations in the GJB2 gene were found in 156 cases (2.80%), in the SLC26A4 gene in 73 cases (1.31%), in the mitochondrial 12S rRNA gene in 10 cases (0.18%), and in the GJB3 gene in 16 cases (0.29%). Additionally, one case of complex heterozygous mutations was detected (0.02%). The positive samples were verified by Sanger sequencing, revealing 9 cases with inconsistent results. Therefore, the accuracy of the fluorescence PCR melting curve method is 96.48%. Conclusion The GJB2 gene and SLC26A4 gene are the main mutations associated with deafness in newborns in this region. Fluorescence PCR fusion curve method combined with Sanger sequencing method accurately detects genetic mutations related to deafness. This mothed is helpful for early detection of congenital, drug-induced, and delayed-onset deafness, playing an important role in the early prevention and treatment of deafness.
- Deafness gene /
- Gene mutation /
- Sanger sequencing /
- Newborn

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图 1 9例不同结果的Sanger测序图

Figure 1. Sanger sequencing images from 9 cases with varied results

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表 1 荧光PCR熔解曲线法检测结果分析

Table 1. Analysis of the detection results using fluorescence PCR melting curve method

基因	突变位点	检出人数(%)
GJB2	c.176-191del16杂合突变	6(0.11)
	c.299-300delAT杂合突变	29(0.52)
	c.235delC纯合突变	1(0.02)
	c.235delC杂合突变	119(2.14)
	c.35delG杂合突变	1(0.02)
GJB3	c.538C＞T	16(0.29)
SLC26A4	c.919-2A＞G纯合突变	1(0.02)
	c.919-2A＞G杂合突变	47(0.84)
	c.2168A＞G杂合突变	8(0.14)
	c.1174A＞T杂合突变	2(0.04)
	c.1226G＞A杂合突变	3(0.05)
	c.1229C＞T杂合突变	6(0.11)
	c.1707+5G＞A杂合突变	4(0.07)
	c.1975G＞C杂合突变	1(0.02)
	c.2027T＞A杂合突变	1(0.02)
线粒体12S rRNA	m.1494C＞T同质性突变	1(0.02)
	m.1555A＞G同质性突变	6(0.11)
	m.1555A＞G异质性突变	3(0.05)
GJB2/线粒体12S rRNA	c.235delC/m.1494C＞T同质性突变	1(0.02)
合计		256(4.60)

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表 2 Sanger测序结果分析

Table 2. Analysis of Sanger sequencing results

基因	荧光PCR熔解曲线法	Sanger测序
GJB2	c.235delC杂合突变	c.232G＞A杂合突变
	c.35delG杂合突变	c.35G＞T杂合突变
SLC26A4	c.1229C＞T杂合突变	c.1233C＞T杂合突变
	c.1229C＞T杂合突变	c.1233C＞T杂合突变
	c.1707+5G＞A	c.1707A＞G杂合突变
	c.1707+5G＞A	c.1707A＞G杂合突变
	c.1707+5G＞A	c.1707A＞G杂合突变
	c.919-2A＞G杂合突变	c.921G＞A杂合突变
	c.919-2A＞G杂合突变	c.919-2A＞G杂合突变；c.1983C＞A杂合突变

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