Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods

Abstract

Porphyromonas gingivalis is an important oral pathogen that causes periodontal disease and is difficult to culture under conventional conditions. Therefore, a reliable technique for detecting this pathogenic bacterium is required. Here, isothermal recombinase polymerase amplification (RPA), a new nucleic acid amplification method, was combined with a visualization method based on nanoparticle-based lateral flow strips (LFS) for the rapid detection of P. gingivalis. The species-specific 16S rRNA sequence of P. gingivalis was used as the target for RPA, and a set of specific primer-probe combinations were designed and screened to amplify the target sequences. As a thermostatic amplification method, the RPA reaction, under optimized conditions, takes only 30 min to complete at a constant temperature (37°C). The amplification reaction products can be detected visually by LFS without any need for special equipment. The RPA-LFS method established for the detection of P. gingivalis was shown to be highly specific in distinguishing P. gingivalis from other pathogenic organisms by using 20 clinical isolates of P. gingivalis and 23 common pathogenic microorganisms. Susceptibility measurements and probit regression analysis were performed with gradient dilutions of P. gingivalis genomic DNA. The method was obtained to be highly sensitive, with a detection limit of 9.27 CFU per reaction at 95% probability. By analyzing the gingival sulcus fluid specimens from 130 patients with chronic periodontitis, the results showed that the RPA-LFS method detected 118 positive cases and 12 negative cases of P. gingivalis, and the results obtained were consistent with those of a conventional PCR assay. The RPA-LFS method is an efficient, rapid, and convenient diagnostic method that simplifies the tedious process of detecting P. gingivalis.

Keywords: Porphyromonas gingivalis; isothermal (DNA) amplification; lateral flow strip; periodontal disease; rapid detection.

Figure 1

Schematic diagram of the RPA-LFS method. (A) Principle of RPA amplification. DNA strands are represented as horizontal lines, and base pairing is represented as short vertical lines between DNA strands. Base pairing is indicated as a short vertical line between DNA strands. (B) Schematic representation of the lateral flow strip (LFS) working principle. The shapes and their representative molecules are listed below the graphic.

Figure 2

RPA primer set screening. The genomic DNA of the P. gingivalis standard strain was used as the template, and amplification with the RPA method was performed with primers set #1–5. The NTC strip is the no-template control for the corresponding RPA reaction, The amplification of the products was confirmed with 1.5% agarose gel electrophoresis.

Figure 3

Performance of the primer–probe combinations tested with the RPA–LFS reaction. The results of LFS detection of the RPA amplification products are shown. The name of each primer–probe set is shown on the corresponding strip. The NTC strip is a template-free control for the individual RPA reaction. The reactions were performed for 30 min at 37°C. The positions of the test and control lines are marked on the right side of the bar graph. The image represents the results of three independent experiments.

Figure 4

Testing the modified primer–probe combinations with the RPA–LFS reaction. (A) Graph shows the LFS results of RPA amplification. (B) The RPA amplification products were analyzed with agarose gel electrophoresis. The name of each primer–probe set is shown above the corresponding strip. The NTC strip is the no-template control for the corresponding RPA reaction, on the strip immediately to its left.

Figure 5

Specificity analysis of the P. gingivalis RPA–LFS assay. The specificity of the RPA–LFS assay established for P. gingivalis was tested on 20 clinical isolates of P. gingivalis (A) and genomic DNA extracted from 20 common pathogenic bacteria (B). No-template control (NTC) was used as the negative control and P. gingivalis as the positive control. RPA amplification results were detected with LFS, and the samples are labeled at the top of the bar graph.

Figure 6

Determination of the limit of detection (LOD) of P. gingivalis RPA–LFS. (A) The LOD for the P. gingivalis RPA-LFS assay system established was determined from 10 independent assays using P. gingivalis genomic DNA at serial dilutions from 6 × 10⁴ to 6 × 10⁻¹ CFU/μL. Images show the results of RPA–LFS, with the amount of template shown at the top of the strip. (B) The group with 10 ng human genomic DNA added in addition to the P. gingivalis genomic DNA. (C) Probit regression analysis of data collected from ten replicates, performed with the SPSS software.