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A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms
INTRODUCTION: The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. MATERIAL AND METHODS: Us...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Sciendo
2020
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305652/ https://www.ncbi.nlm.nih.gov/pubmed/32587912 http://dx.doi.org/10.2478/jvetres-2020-0033 |
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author | Zhang, Shi-Jun Wang, Lu-Lu Lu, Shi-Ying Hu, Pan Li, Yan-Song Zhang, Ying Chang, Heng-Zhen Zhai, Fei-Fei Liu, Zeng-Shan Li, Zhao-Hui Ren, Hong-Lin |
author_facet | Zhang, Shi-Jun Wang, Lu-Lu Lu, Shi-Ying Hu, Pan Li, Yan-Song Zhang, Ying Chang, Heng-Zhen Zhai, Fei-Fei Liu, Zeng-Shan Li, Zhao-Hui Ren, Hong-Lin |
author_sort | Zhang, Shi-Jun |
collection | PubMed |
description | INTRODUCTION: The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. MATERIAL AND METHODS: Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. RESULTS: The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R(2)) of the standard curve was 0.999. The sensitivity of the method was 10(3) CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. CONCLUSION: In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research. |
format | Online Article Text |
id | pubmed-7305652 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Sciendo |
record_format | MEDLINE/PubMed |
spelling | pubmed-73056522020-06-24 A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms Zhang, Shi-Jun Wang, Lu-Lu Lu, Shi-Ying Hu, Pan Li, Yan-Song Zhang, Ying Chang, Heng-Zhen Zhai, Fei-Fei Liu, Zeng-Shan Li, Zhao-Hui Ren, Hong-Lin J Vet Res Review Article INTRODUCTION: The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. MATERIAL AND METHODS: Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. RESULTS: The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R(2)) of the standard curve was 0.999. The sensitivity of the method was 10(3) CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. CONCLUSION: In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research. Sciendo 2020-05-12 /pmc/articles/PMC7305652/ /pubmed/32587912 http://dx.doi.org/10.2478/jvetres-2020-0033 Text en © 2020 S.J. Zhang et al. published by Sciendo http://creativecommons.org/licenses/by-nc-nd/3.0 This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. |
spellingShingle | Review Article Zhang, Shi-Jun Wang, Lu-Lu Lu, Shi-Ying Hu, Pan Li, Yan-Song Zhang, Ying Chang, Heng-Zhen Zhai, Fei-Fei Liu, Zeng-Shan Li, Zhao-Hui Ren, Hong-Lin A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms |
title | A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms |
title_full | A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms |
title_fullStr | A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms |
title_full_unstemmed | A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms |
title_short | A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms |
title_sort | novel, rapid, and simple pma-qpcr method for detection and counting of viable brucella organisms |
topic | Review Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305652/ https://www.ncbi.nlm.nih.gov/pubmed/32587912 http://dx.doi.org/10.2478/jvetres-2020-0033 |
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