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Enzyme-free and sensitive method for single-stranded nucleic acid detection based on CHA and HCR
Xiaolong Chen1, Chaowang Huang1, Fuping Nie2
1Department of Geriatrics and Special Services Medicine, Xinqiao Hospital, Army Military Medical University, Chongqing 400037, China. humingdong@tmmu.edu.cn.
This study introduces an enzyme-free method for detecting single-stranded nucleic acids using hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA). This sensitive approach offers rapid and simple detection, ideal for clinical applications like foot-and-mouth disease virus (FMDV) screening.
Area of Science:
- Biochemistry
- Molecular Biology
- Diagnostics
Background:
- Enzyme-free nucleic acid detection methods are crucial for simplifying clinical testing.
- Current methods often rely on enzymes, increasing complexity and cost.
- Sensitive and rapid detection of single-stranded nucleic acids is a significant challenge.
Purpose of the Study:
- To develop a novel, enzyme-free method for sensitive single-stranded nucleic acid detection.
- To combine hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) for enhanced detection.
- To create a simplified and rapid diagnostic tool for clinical applications.
Main Methods:
- An enzyme-free assay was designed utilizing HCR and CHA triggered by target nucleic acids.
- Auxiliary hairpin strands were engineered to expose sequences for CHA and HCR initiation.
- 2-aminopurines were incorporated for fluorescence-based signal generation without additional reporters.
Main Results:
- The combined HCR-CHA method demonstrated a nearly 10-fold increase in sensitivity compared to individual methods.
- The assay achieved specific detection of foot-and-mouth disease virus (FMDV) down to 2.78 × 10^1 TCID50.
- The method showed improved target recognition and effective FMDV detection.
Conclusions:
- The developed enzyme-free HCR-CHA method provides a simple, rapid, and sensitive approach for single-stranded nucleic acid detection.
- This technique holds potential for primary screening of viruses like FMDV, especially in resource-limited settings.
- The method offers advantages in speed and simplicity over traditional techniques like RT-qPCR for certain applications.
