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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...

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Autores principales: Tak, Lee-Jung, Shin, Min-Kyoung, Yoo, Jun-Il, Cho, Min-Chul, Kim, Wanil
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
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.
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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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