Application of the real-time fluorescence PCR melting curve method in gene screening of non-syndromic hearing loss
10.3760/cma.j.issn.1673-0860.2019.04.009
- VernacularTitle: 实时荧光PCR熔解曲线法在非综合征型耳聋基因检测中的应用研究
- Author:
Yalan LIU
1
,
2
;
Xiaohong JIANG
1
,
2
;
Jie SUN
3
;
Lingyun MEI
1
,
2
;
Chufeng HE
1
,
2
;
Yuyuan DENG
1
,
2
;
Jie WEN
1
,
2
;
Yong FENG
1
,
2
Author Information
1. Department of Otorhinolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha 410008, China
2. Province Key Laboratory of Otorhinolaryngology Critical Diseases, Changsha 410008, China
3. Department of Otorhinolaryngology Head and Neck Surgery, Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen 518033, China
- Publication Type:Journal Article
- Keywords:
Hearing loss, sensorineural;
Genetic diseases, inborn;
Polymerase chain reaction;
DNA mutational analysis
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2019;54(4):286-291
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To detect 20 common deafness gene mutations in non-syndromic hearing loss patients in China using the melting curve method, and analyze and summarize the mutation data to explore the clinical value of this method.
Methods:The real-time fluorescence PCR melting curve method was used to detect 20 common mutations of four deafness genes(GJB2,GJB3,SLC26A4 and mtDNA) in 492 patients with non-syndromic hearing loss recruited between March 2014 and September 2016 from the Otolaryngology Department of Xiangya Hospital, Central South University(283 males and 209 females, the age ranged from 1 to 48 years old). The Sanger sequencing method was used to compare the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the deafness mutation detected by the real-time fluorescence PCR melting curve method.
Results:A total of 492 samples were detected. 193 wild-type samples, 93 homozygous mutant samples, 145 heterozygous mutant samples, 59 composite heterozygous mutant samples and 2 samples with unknown mutations were detected using the real-time fluorescence PCR melting curve method within the range of 20 gene mutations, whichwere identical to the Sanger sequencing results.The two samples were detected as unknown mutations by the real-time fluorescent PCR melting curve method were confirmed by Sanger sequencing, including a composite heterozygous mutant sample and a homogenous mutation sample. GJB2 c.235delC and SLC26A4 c.919-2 A>G were the most common hotspot mutations in this study, followed by mtDNA m.1555 A>G. Compared with the Sanger sequencing method, the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the real-time fluorescence PCR melting curve method were 100%, the Youden′s index was 1.0, and the Kappa value was 1.
Conclusions:The real-time fluorescence PCR melting curve method is suitable for the detection of deafness gene mutations. It has the advantages in terms of simple, rapid, high sensitivity and strong specificity and can accurately detect the 20 gene mutations of 4 common deafness genes in Chinese population, which is expected to be used for the clinical detection of deafness genes in the future.
