Establishment and application of a rapid molecular diagnostic platform for the isothermal visual amplification of group B Streptococcus based on recombinase polymerase

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.

Keywords: cfb; group B streptococcus; isothermal amplification; molecular diagnostic methods; recombinase polymerase.

Figure 1

Schematic of RPA amplification and LFS visualization.

Figure 2

Results of cfb gene-specific RPA primers and probe combination screening. The cfb gene of GBS was amplified by RPA and detected by LFS visualization with combinations of cfb-F1/R1/P, cfb-F1/R2/P, cfb-F2/R1/P, and cfb-F2/R2/P. Each combination is identified above its corresponding LFS, which is marked by a control line (C) and a detection line (T). The experimental group that used nuclease-free water instead of a template served as the negative control (NTC).

Figure 3

Screening results of the optimal reaction temperature and time of the GBS detection method using RPA-LFS. (A) The cfb-F2/R1/P primer and probe combination was used for reactions at 16°C, 25°C, 30°C, 37°C, 39°C, 42°C, 50°C, and 60°C, each for 30 min. The optimal reaction temperature was screened by LFS detection. (B) The reaction times were 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and 45 min at 39°C, and the optimum reaction time was screened by LFS detection.

Figure 4

Interspecific detection results of the RPA-LFS-based GBS detection method. Thermally cleaved genomic DNA of (A) Streptococcus agalactiae (ATCC 27956 and 8 isolates) and (B) L. iners, L. crispatus, C. albicans, L. monocytogenes, Y. enterocolitica, K. Pneumoniae, E. cloacae, C. freundii, V. alginolyticus, V. parahaemolyticus, S. typhimurium, S. aureus, P. aeruginosa, T. vaginalis were used as templates. A no template control (NTC) was also established. The RPA-LFS-based GBS detection method was conducted under 39°C for 30 min, and the results were detected by LFS.

Figure 5

Results of detection sensitivity for the GBS detection method based on RPA-LFS. (A) The RPA-LFS results were obtained by using crude S. agalactiae solutions as the template, with final concentrations ranging from 10⁵ copies/μL to 10¹ copies/μL. (B) The RPA-LFS results were determined by using crude S. agalactiae spiked urine as template with concentrations from 10⁵ copies/μL to 10¹ copies/μL. (C) Detection results of RPA-LFS using genomic DNA purified from S. agalactiae-spiked urine, with final concentrations ranging from 10⁵ copies/μL to 10¹ copies/μL as a template.