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Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid
Periprosthetic joint infection (PJI) can be diagnosed to characterize the microorganisms constituting a biofilm, which is an essential procedure for proper treatment. The gold standard method for detecting and identifying the causative microorganism is culture of microorganisms from patients-derived...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10651726/ https://www.ncbi.nlm.nih.gov/pubmed/38029245 http://dx.doi.org/10.3389/fcimb.2023.1301446 |
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author | Tak, Lee-Jung Shin, Min-Kyoung Yoo, Jun-Il Cho, Min-Chul Kim, Wanil |
author_facet | Tak, Lee-Jung Shin, Min-Kyoung Yoo, Jun-Il Cho, Min-Chul Kim, Wanil |
author_sort | Tak, Lee-Jung |
collection | PubMed |
description | Periprosthetic joint infection (PJI) can be diagnosed to characterize the microorganisms constituting a biofilm, which is an essential procedure for proper treatment. The gold standard method for detecting and identifying the causative microorganism is culture of microorganisms from patients-derived sample.; however, this method takes a long time and has low sensitivity. To compensate for these limitations, identification methods based on real-time PCR (RT-PCR) have been widely used. However, RT-PCR also has limitations, including low sensitivity and the requirement of a standard curve for quantification. Therefore, to prevent significant proliferation of pathogenic bacteria, it is important to detect a limited number of infectious bacteria during early stages of PJI. In the present study, we developed droplet digital PCR-based detection of bacterial pathogens in PJI. And we evaluated the analytical performance of the assay using a model plasmid, based on the 16S ribosomal DNA sequence of target bacteria commonly found in PJI. We also prepared genomic DNA extracted from E. coli, S. aureus, and S. epidermidis to test whether ddPCR provides better sensitivity and quantification of the target sequences. ddPCR detected 400 attograms of target DNA, which was more than 10 times less than that detected by real-time PCR using synthesized plasmid. In addition, ddPCR detected target regions from genomic DNA of 50 femtograms for E. coli, 70 femtograms for S. epidermidis, and 90 femtograms for S. aureus. The results indicate that ddPCR has the potential to decrease the microbial detection limit and provide precise detection, signifying its effectiveness for early PJI. |
format | Online Article Text |
id | pubmed-10651726 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-106517262023-01-01 Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid Tak, Lee-Jung Shin, Min-Kyoung Yoo, Jun-Il Cho, Min-Chul Kim, Wanil Front Cell Infect Microbiol Cellular and Infection Microbiology Periprosthetic joint infection (PJI) can be diagnosed to characterize the microorganisms constituting a biofilm, which is an essential procedure for proper treatment. The gold standard method for detecting and identifying the causative microorganism is culture of microorganisms from patients-derived sample.; however, this method takes a long time and has low sensitivity. To compensate for these limitations, identification methods based on real-time PCR (RT-PCR) have been widely used. However, RT-PCR also has limitations, including low sensitivity and the requirement of a standard curve for quantification. Therefore, to prevent significant proliferation of pathogenic bacteria, it is important to detect a limited number of infectious bacteria during early stages of PJI. In the present study, we developed droplet digital PCR-based detection of bacterial pathogens in PJI. And we evaluated the analytical performance of the assay using a model plasmid, based on the 16S ribosomal DNA sequence of target bacteria commonly found in PJI. We also prepared genomic DNA extracted from E. coli, S. aureus, and S. epidermidis to test whether ddPCR provides better sensitivity and quantification of the target sequences. ddPCR detected 400 attograms of target DNA, which was more than 10 times less than that detected by real-time PCR using synthesized plasmid. In addition, ddPCR detected target regions from genomic DNA of 50 femtograms for E. coli, 70 femtograms for S. epidermidis, and 90 femtograms for S. aureus. The results indicate that ddPCR has the potential to decrease the microbial detection limit and provide precise detection, signifying its effectiveness for early PJI. Frontiers Media S.A. 2023-11-02 /pmc/articles/PMC10651726/ /pubmed/38029245 http://dx.doi.org/10.3389/fcimb.2023.1301446 Text en Copyright © 2023 Tak, Shin, Yoo, Cho and Kim 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 Tak, Lee-Jung Shin, Min-Kyoung Yoo, Jun-Il Cho, Min-Chul Kim, Wanil Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
title | Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
title_full | Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
title_fullStr | Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
title_full_unstemmed | Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
title_short | Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
title_sort | development of droplet digital pcr-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid |
topic | Cellular and Infection Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10651726/ https://www.ncbi.nlm.nih.gov/pubmed/38029245 http://dx.doi.org/10.3389/fcimb.2023.1301446 |
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