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

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.

Idioma:: Inglés

Calendario de la edición:: 4 veces al año
Temas de la revista:: Ciencias de la vida, Biología molecular, Microbiología y virología, Ciencias de la vida, otros, Medicina, Medicina veterinaria

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

Publicado en línea: 12 may 2020

Páginas: 253 - 261

Recibido: 19 sept 2019

Aceptado: 28 abr 2020

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

Palabras claveviable 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

Material and Methods

Results

Conclusion

Palabras clave
viable bacteria, gene, propidium monoazide, quantitative PCR