Open AccessA novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms
Author(s) -
Shijun Zhang,
Lulu Wang,
Shiying Lu,
Pan Hu,
Yansong Li,
Ying Zhang,
Heng-Zhen Chang,
Fei-Fei Zhai,
Zengshan Liu,
Zhaohui Li,
Hong-Lin Ren
Publication year - 2020
Publication title -
journal of veterinary research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.469
H-Index - 25
eISSN - 2450-8608
pISSN - 2450-7393
DOI - 10.2478/jvetres-2020-0033
Subject(s) - repeatability , brucella , biology , propidium monoazide , standard curve , real time polymerase chain reaction , cell counting , microbiology and biotechnology , serial dilution , recombinant dna , chromatography , gene , chemistry , virology , brucellosis , genetics , medicine , biochemistry , alternative medicine , pathology , cell cycle
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.