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Updated: Apr 12, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Rapid identification of aminoglycoside-induced deafness gene mutations using multiplex real-time polymerase chain
Shasha Huang1, Guangxin Xiang2, Dongyang Kang1
1Department of Otolaryngology, PLA General Hospital, Do. 28 Fuxing Rode, Beijing 100853, People's Republic of China.
Background:
Exposure to aminoglycoside antibiotics can induce ototoxicity in genetically susceptible individuals carrying certain mitochondrial DNA (mtDNA) mutations (C1494T and A1555G), resulting in hearing loss. So, a rapid diagnostic approach is needed to accurately identify subjects carrying such gene mutations.
Methods:
In the present study, we describe a rapid and reliable four-color, real-time quantitative polymerase chain reaction (qPCR) assay for simultaneously detecting two mtDNA 12S rRNA gene variants, A1555G and C1494T, which are prevalent in the Han Chinese population. This multiplex assay incorporates three allele-specific TaqMan probes labeled with different fluorophores in a single reaction, providing high genotyping accuracy for clinical blood samples.
Results:
Tests with C1494T, A1555G and wild-type DNA exhibited high sensitivity, specificity, reproducibility and accuracy of discriminating mutations from wild-type.
Conclusions:
This study shows that this simple and inexpensive method can be used for routine molecular diagnostics and potentially for large-scale genetic screening.
Insights
A new real-time PCR assay rapidly identifies mitochondrial DNA (mtDNA) mutations (A1555G and C1494T) linked to antibiotic-induced hearing loss. This accurate and inexpensive method aids in genetic screening for ototoxicity risk.
Area of Science:
- Genetics
- Molecular Biology
- Pharmacogenomics
Background:
- Aminoglycoside antibiotics can cause ototoxicity (hearing loss) in individuals with specific mitochondrial DNA (mtDNA) mutations.
- Mitochondrial DNA mutations A1555G and C1494T are known genetic factors predisposing to this hearing loss.
- Accurate and rapid identification of these mutations is crucial for preventing ototoxicity.
Purpose of the Study:
- To develop and validate a rapid, multiplex, real-time quantitative PCR (qPCR) assay.
- To simultaneously detect two common mtDNA variants (A1555G and C1494T) associated with aminoglycoside-induced ototoxicity.
- To provide a reliable diagnostic tool for clinical settings.
Main Methods:
- A four-color, real-time qPCR assay was designed.
- The assay utilizes three allele-specific TaqMan probes for simultaneous detection of A1555G and C1494T mutations.
- The method was validated using clinical blood samples.
Main Results:
- The developed multiplex qPCR assay demonstrated high sensitivity, specificity, reproducibility, and accuracy.
- The assay reliably distinguished between mutant and wild-type mtDNA sequences.
- Genotyping accuracy was high for clinical blood samples.
Conclusions:
- The described qPCR assay is a simple, inexpensive, and effective method for detecting specific mtDNA mutations.
- This assay is suitable for routine molecular diagnostics.
- The method holds potential for large-scale genetic screening to identify individuals at risk of ototoxicity.
