Cargando…

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

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 nu...

Descripción completa

Detalles Bibliográficos
Autores principales: Ge, Duobao, Wang, Fang, Hu, Yanyan, Wang, Bendi, Gao, Xuzhu, Chen, Zhenxing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9174636/
https://www.ncbi.nlm.nih.gov/pubmed/35694545
http://dx.doi.org/10.3389/fcimb.2022.895261
_version_ 1784722281450176512
author Ge, Duobao
Wang, Fang
Hu, Yanyan
Wang, Bendi
Gao, Xuzhu
Chen, Zhenxing
author_facet Ge, Duobao
Wang, Fang
Hu, Yanyan
Wang, Bendi
Gao, Xuzhu
Chen, Zhenxing
author_sort Ge, Duobao
collection PubMed
description 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.
format Online
Article
Text
id pubmed-9174636
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-91746362022-06-09 Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods Ge, Duobao Wang, Fang Hu, Yanyan Wang, Bendi Gao, Xuzhu Chen, Zhenxing Front Cell Infect Microbiol Cellular and Infection Microbiology 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. Frontiers Media S.A. 2022-05-25 /pmc/articles/PMC9174636/ /pubmed/35694545 http://dx.doi.org/10.3389/fcimb.2022.895261 Text en Copyright © 2022 Ge, Wang, Hu, Wang, Gao and Chen https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Ge, Duobao
Wang, Fang
Hu, Yanyan
Wang, Bendi
Gao, Xuzhu
Chen, Zhenxing
Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods
title Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods
title_full Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods
title_fullStr Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods
title_full_unstemmed Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods
title_short Fast, Simple, and Highly Specific Molecular Detection of Porphyromonas gingivalis Using Isothermal Amplification and Lateral Flow Strip Methods
title_sort fast, simple, and highly specific molecular detection of porphyromonas gingivalis using isothermal amplification and lateral flow strip methods
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9174636/
https://www.ncbi.nlm.nih.gov/pubmed/35694545
http://dx.doi.org/10.3389/fcimb.2022.895261
work_keys_str_mv AT geduobao fastsimpleandhighlyspecificmoleculardetectionofporphyromonasgingivalisusingisothermalamplificationandlateralflowstripmethods
AT wangfang fastsimpleandhighlyspecificmoleculardetectionofporphyromonasgingivalisusingisothermalamplificationandlateralflowstripmethods
AT huyanyan fastsimpleandhighlyspecificmoleculardetectionofporphyromonasgingivalisusingisothermalamplificationandlateralflowstripmethods
AT wangbendi fastsimpleandhighlyspecificmoleculardetectionofporphyromonasgingivalisusingisothermalamplificationandlateralflowstripmethods
AT gaoxuzhu fastsimpleandhighlyspecificmoleculardetectionofporphyromonasgingivalisusingisothermalamplificationandlateralflowstripmethods
AT chenzhenxing fastsimpleandhighlyspecificmoleculardetectionofporphyromonasgingivalisusingisothermalamplificationandlateralflowstripmethods