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Establishment and application of a rapid molecular diagnostic platform for the isothermal visual amplification of
Meilin Liu1, Huan Wang1, Chu Chu1
1Obstetrical Department, Lianyungang Maternal and Child Health Hospital, Lianyungang, Jiangsu, China.
Insights
A new visual detection method using recombinase polymerase amplification (RPA) and lateral flow strip (LFS) can rapidly screen for Group B Streptococcus (GBS) infections in pregnant women. This fast, sensitive, and device-independent approach offers a feasible strategy for clinical GBS detection.
Area of Science:
- Microbiology
- Medical Diagnostics
- Biotechnology
Background:
- Group B streptococcal (GBS) infections pose significant risks during perinatal pregnancies, including premature delivery, neonatal septicemia, and meningitis.
- Urgent need for rapid, sensitive, and accessible GBS screening methods throughout pregnancy.
Purpose of the Study:
- To develop a device-independent, fast, sensitive, and visual method for detecting Group B Streptococcus (GBS).
- To utilize recombinase polymerase isothermal amplification (RPA) and lateral flow chromatography strip (LFS) for GBS detection.
Main Methods:
- Designed primers and a probe targeting the GBS cfb gene.
- Optimized RPA reaction conditions (temperature, time) and primer/probe combinations.
- Validated the GBS-RPA-LFS method using vaginal swab samples from pregnant women, assessing specificity and detection limits.
Main Results:
- The GBS-RPA-LFS method detected GBS within 15 minutes at 39-42°C.
- Demonstrated high specificity, with no cross-reactivity against common vaginal pathogens.
- Achieved a detection limit of 100 copies per reaction and 95.92% agreement with TaqMan real-time PCR in clinical samples.
Conclusions:
- Successfully established a combined RPA and LFS platform for rapid, sensitive, and specific GBS detection.
- The developed method is portable, device-independent, and suitable for clinical GBS screening in perinatal pregnancies.
- Offers a feasible and efficient strategy to mitigate adverse pregnancy outcomes associated with GBS.
Abstract:
With growing concerns about Group B streptococcal (GBS) infections and their adverse effects on perinatal pregnancies, including infection, premature delivery, neonatal septicemia, and meningitis, it is urgent to promote GBS screening at all pregnancy stages. The purpose of this study is to establish a device-independent, fast, sensitive, and visual GBS detection method. Taking advantage of the characteristics of the recombinase polymerase isothermal amplification (RPA), the activity of the nfo nuclease cleavage base analog (tetrahydrofuran, THF) site, and the advantages of visual reading of the lateral flow chromatography strip (LFS), a GBS detection method was developed. This method focused on the conservative region of the Christie-Atkins-Munch-Petersen factor encoded by the cfb gene, a virulence gene specific to GBS. Two forward primers, two biotin-labeled reverse primers, and one fluorescein isothiocyanate (FITC)-labeled and C3spacer-blocked probe were designed. The study involved optimizing the primer pair and probe combination, determining the optimal reaction temperature and time, evaluating specificity, analyzing detection limits, and testing the method on 87 vaginal swabs from perinatal pregnant women. The results showed that the visual detection method of GBS-RPA-LFS, using the cfb-F1/R2/P1 primer probe, could detect GBS within 15 min at the temperature ranging from 39°C to 42°C. Furthermore, the method specifically amplified only GBS, without cross-reacting with pathogens like Lactobacillus iners, Lactobacillus crispatus, Candida albicans, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginolyticus, Vibrio parahaemolyticus, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, or Trichomonas vaginalis. It could detect a minimum of 100 copies per reaction. In clinical 98 samples of vaginal swabs from pregnant women, the agreement rate between the GBS-RPA-LFS method and TaqMan real-time fluorescence quantification method was 95.92%. In conclusion, this study successfully established a combined RPA and LFS GBS in situ detection platform, with short reaction time, high sensitivity, high specificity, portability, and device independence, providing a feasible strategy for clinical GBS screening.
