VGRCOT: a one-tube visual detection method for group B Streptococcus combining RPA and CRISPR/Cas12a for point-of-care testing in reproductive health.

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.