A novel, rapid, and simple PMA-qPCR method for detection and counting of viable  organisms

Shi-Jun Zhang; Lu-Lu Wang; Shi-Ying Lu; Pan Hu; Yan-Song Li; Ying Zhang; Heng-Zhen Chang; Fei-Fei Zhai; Zeng-Shan Liu; Zhao-Hui Li; Hong-Lin Ren

Open Access

A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms

Journal of Veterinary Research

Volume 64 (2020): Issue 2 (June 2020)

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Page range: 253 - 261

Received: Sep 19, 2019

Accepted: Apr 28, 2020

DOI: https://doi.org/10.2478/jvetres-2020-0033

Keywords
viable bacteria, gene, propidium monoazide, quantitative PCR

© 2020 S.J. Zhang et al. published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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 (R²) of the standard curve was 0.999. The sensitivity of the method was 10³ 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.

eISSN:: 2450-8608
Language:: English

Publication timeframe:: 4 times per year
Journal Subjects:: Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine

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