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

Shi-Jun Zhang

Shi-Jun Zhang

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Zhang, Shi-Jun
, 
Lu-Lu Wang

Lu-Lu Wang

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Wang, Lu-Lu
, 
Shi-Ying Lu

Shi-Ying Lu

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Lu, Shi-Ying
, 
Pan Hu

Pan Hu

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Hu, Pan
, 
Yan-Song Li

Yan-Song Li

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Li, Yan-Song
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Ying Zhang

Ying Zhang

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Zhang, Ying
, 
Heng-Zhen Chang

Heng-Zhen Chang

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Chang, Heng-Zhen
, 
Fei-Fei Zhai

Fei-Fei Zhai

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Zhai, Fei-Fei
, 
Zeng-Shan Liu

Zeng-Shan Liu

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Liu, Zeng-Shan
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Zhao-Hui Li

Zhao-Hui Li

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Li, Zhao-Hui
 and 
Hong-Lin Ren

Hong-Lin Ren

Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, College of Animal Sciences, Jilin UniversityChangchun, China
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Ren, Hong-Lin
  
May 12, 2020
A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms's Cover Image
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Published Online: May 12, 2020
Page range: 253 - 261
Received: Sep 19, 2019
Accepted: Apr 28, 2020
DOI: https://doi.org/10.2478/jvetres-2020-0033
Keywords
© 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.

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