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VGRCOT: a one-tube visual detection method for group B Streptococcus combining RPA and CRISPR/Cas12a for
Caixia Ji1, Liqiang Ru2,3, Tiao Han4
1Department of Clinical Laboratory, People's Hospital of Deyang City, Chengdu University of Traditional Chinese Medicine, Deyang, Sichuan, China.
Insights
A new visual nucleic acid detection method, VGRCOT, combines RPA and CRISPR/Cas12a for rapid Group B Streptococcus (GBS) screening in pregnant women. This sensitive, one-tube assay offers a promising tool for point-of-care diagnostics to prevent neonatal infections.
Area of Science:
- Molecular Diagnostics
- Microbiology
- Biotechnology
Background:
- Group B Streptococcus (GBS) poses a significant threat to pregnant women and newborns, necessitating effective screening and prophylactic measures.
- Current diagnostic methods for GBS can be time-consuming or require specialized equipment, limiting their use in resource-limited settings.
- Rapid and accurate detection of GBS is crucial for timely intervention and prevention of serious perinatal infections.
Purpose of the Study:
- To develop a novel, visual, one-tube nucleic acid detection method for Group B Streptococcus (GBS).
- To integrate recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology for enhanced sensitivity and specificity.
- To establish a convenient and accurate diagnostic tool for GBS screening, suitable for point-of-care applications.
Main Methods:
- Development of a visual nucleic acid method (VGRCOT) combining RPA and CRISPR/Cas12a in a single tube.
- Optimization of reagent concentrations (ssDNA-FQ reporter, crRNA) and reaction times (RPA, CRISPR/Cas12a cleavage).
- Evaluation of VGRCOT's sensitivity, selectivity, and performance against qPCR using clinical specimens.
Main Results:
- The VGRCOT method achieved visual detection of GBS through fluorescence under UV light.
- Optimal conditions yielded a detection limit of 10^1 copies/reaction with satisfactory selectivity.
- VGRCOT demonstrated comparable performance to qPCR in detecting GBS in clinical samples.
- The one-tube design with reagents in the tube bottom and lid simplified operation and prevented aerosol contamination.
Conclusions:
- VGRCOT offers a sensitive, specific, and visually detectable method for GBS identification.
- The assay's ease of use, rapid detection (within 60 min), and suitability for resource-limited settings make it ideal for point-of-care testing.
- The VGRCOT platform's adaptability through editable crRNA supports potential for detecting other pathogens, enabling scalable and cost-effective diagnostics.
Abstract:
Group B Streptococcus (GBS) is a significant pathogen that causes perinatal infections, seriously threatening the health of pregnant women and newborns. Prophylactic antibiotic treatment for pregnant women who screen positive for GBS can notably reduce the incidence and fatality of neonatal infections. Herein, we developed a visual nucleic acid method for GBS that integrates RPA and CRISPR/Cas12a in a one-tube setup, termed VGRCOT. The VGRCOT method achieved one-tube detection by adding the appropriate reagents to the bottom and lid of the EP tube, respectively. By rigorous optimization of ssDNA-FQ reporter concentration, crRNA concentration, RPA reaction time, and CRISPR/Cas12a cleavage time, VGRCOT can exhibit fluorescence under ultraviolet light, enabling visual detection. Under optimal conditions, VGRCOT has a satisfactory selectivity, and the detection limit was determined as 101 copies/reaction. Finally, VGRCOT also showed good performance comparable to qPCR in the actual detection of clinical specimens. Due to its ease of operation and convenient signal acquisition, VGRCOT shows promise for point-of-care testing in reproductive health.IMPORTANCEThis study presents a convenient, sensitive, and accurate visual detection method (VGRCOT) for GBS, combining RPA and CRISPR/Cas12a in a single reaction vessel. Through optimization of experimental conditions, VGRCOT enables detection within 60 min, with a minimum detection limit of 101 copies per reaction. VGRCOT offers several advantages by adding the appropriate reagents to the bottom and lid of the EP tube. The one-tube visualization method effectively prevents aerosol contamination, simplifies procedures, and enables visual detection without complex instruments, making it ideal for resource-limited environments. Additionally, its editable crRNA and the use of commonly available laboratory reagents allow for easy reprogramming to detect various pathogens, supporting scalable and low-cost batch production.
