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Development of the CRISPR-Cas12a-Based Biosensing System for Rapid, Ultrasensitive, and Highly Specific Detection of
Xinbei Jia1, Yiqin Zhang1, Lin Zhou2
1Department of Otorhinolaryngology Head and Neck Surgery, Children's Hospital Capital Institute of Pediatrics, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100020, China.
Insights
A new diagnostic platform, GAS-MCDA-CRISPR, rapidly and accurately detects Group A Streptococcus (GAS) infections. This highly sensitive test is crucial for timely antibiotic treatment of bacterial pharyngitis in children and adolescents.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Group A Streptococcus (GAS) is a primary cause of bacterial pharyngitis in pediatric populations.
- Accurate and swift diagnosis of GAS is critical for effective antibiotic therapy and preventing complications.
Purpose of the Study:
- To develop and validate a rapid, sensitive, and specific diagnostic platform for GAS detection.
- To establish a reliable tool for identifying GAS infections in clinical settings.
Main Methods:
- Development of the GAS-MCDA-CRISPR assay combining multiple cross displacement amplification (MCDA) and CRISPR-Cas12a.
- Utilizing MCDA to preamplify the GAS-specific *speB* gene.
- Employing a CRISPR-Cas12a biosensing system with a fluorescent ssDNA reporter for detection.
Main Results:
- The GAS-MCDA-CRISPR assay achieved a detection limit as low as 45 fg/µL of GAS genomic DNA.
- The assay demonstrated high specificity, with no cross-reactivity against common non-GAS pathogens.
- Successful detection of GAS in 56 clinical samples, confirming its diagnostic accuracy.
Conclusions:
- The GAS-MCDA-CRISPR assay is a highly reliable and rapid diagnostic tool for GAS infections.
- This platform offers a promising solution for timely and accurate diagnosis of bacterial pharyngitis.
- The developed assay can significantly aid in guiding appropriate antibiotic treatment decisions.
Abstract:
Streptococcus pyogenes (group A streptococcus, GAS) is the leading bacterial cause of acute pharyngitis in children and adolescents. Rapid and reliable diagnosis of GAS pharyngitis is essential for guiding a timely antibiotic treatment. Here, we developed a rapid, highly sensitive, and specific test platform for the detection of GAS, designated GAS-MCDA-CRISPR. In this diagnostic platform, the multiple cross displacement amplification (MCDA) technique was utilized to preamplify the specific speB gene of GAS. Subsequently, the CRISPR-Cas12a-based biosensing system was employed to decode the MCDA products. MCDA primers, a guide RNA (gRNA), and a quenched fluorescent single-stranded DNA (ssDNA) reporter were designed to target the speB gene of GAS. The GAS-MCDA-CRISPR assay demonstrated the ability to detect GAS genomic DNA at a concentration as low as 45 fg per microliter while exhibiting no cross-reactivity with other non-GAS pathogens. Moreover, 56 clinical samples were correctly detected by the GAS-MCDA-CRISPR assay. These data highlighted that the GAS-MCDA-CRISPR assay is a reliable diagnostic tool for the reliable and quick diagnosis of GAS infection.
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