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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
, 
Lu-Lu Wang
Lu-Lu Wang
, 
Shi-Ying Lu
Shi-Ying Lu
, 
Pan Hu
Pan Hu
, 
Yan-Song Li
Yan-Song Li
, 
Ying Zhang
Ying Zhang
, 
Heng-Zhen Chang
Heng-Zhen Chang
, 
Fei-Fei Zhai
Fei-Fei Zhai
, 
Zeng-Shan Liu
Zeng-Shan Liu
, 
Zhao-Hui Li
Zhao-Hui Li
 and 
Hong-Lin Ren
Hong-Lin Ren
   | May 12, 2020
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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.

Fig. 1

The target BCSP31 fragment of PCR amplification from Brucella suis S2 strain. M – DL2000 DNA Marker (TaKaRa Bio); lane 1 – DNA fragment of PCR amplification
The target BCSP31 fragment of PCR amplification from Brucella suis S2 strain. M – DL2000 DNA Marker (TaKaRa Bio); lane 1 – DNA fragment of PCR amplification

Fig. 2

The standard curve for calculating Brucella concentration according to CT of qPCR based on the template of recombinant standard plasmid pMD-18T-BCSP31
The standard curve for calculating Brucella concentration according to CT of qPCR based on the template of recombinant standard plasmid pMD-18T-BCSP31

Fig. 3

Optimisation of PMA treatment conditions. A – Effect of different exposure time on qPCR amplification; B – Different concentrations of PMA treating dead bacteria; C – Different concentrations of PMA treating viable bacteria; * – P < 0.05; Yellow columns – optimal PMA treatment time of 10 min; Yellow columns with ☆ – optimal PMA treatment concentration of 15 μg/mL; × – not detected
Optimisation of PMA treatment conditions. A – Effect of different exposure time on qPCR amplification; B – Different concentrations of PMA treating dead bacteria; C – Different concentrations of PMA treating viable bacteria; * – P < 0.05; Yellow columns – optimal PMA treatment time of 10 min; Yellow columns with ☆ – optimal PMA treatment concentration of 15 μg/mL; × – not detected

Fig. 4

The sensitivity of detecting B. suis S2 strain. A – sensitivity by the PMA-qPCR; B – sensitivity by the conventional PCR, comprising M – DL2000 DNA Marker; lanes 1–10 – 108 – 10-1 CFU/mL
The sensitivity of detecting B. suis S2 strain. A – sensitivity by the PMA-qPCR; B – sensitivity by the conventional PCR, comprising M – DL2000 DNA Marker; lanes 1–10 – 108 – 10-1 CFU/mL

Fig. 5

Analysis of the specificity of PMA-qPCR. A – amplification plot; B – melting curves of the qPCR amplification product; C – agarose gel electrophoresis of different Brucella species detected by the normal PCR, comprising M – DL5000 DNA Marker; lane 1 – B. suis S2; lane 2 – B. abortus 2308; lane 3 – B. abortus A19; lane 4 – B. melitensis M5; lane 5 – B. melitensis 16M; and lane 6 – B. ovis; D – agarose gel electrophoresis of species other than Brucella amplified by the conventional PCR, comprising M – DL2000 DNA Marker; lane 1 – B. suis S2; lane 2 – E. coli; lane 3 – S. typhimurium; lane 4 – Y. enterocolitica; lane 5 – V. parahaemolyticus; and lane 6 – RNase-free water
Analysis of the specificity of PMA-qPCR. A – amplification plot; B – melting curves of the qPCR amplification product; C – agarose gel electrophoresis of different Brucella species detected by the normal PCR, comprising M – DL5000 DNA Marker; lane 1 – B. suis S2; lane 2 – B. abortus 2308; lane 3 – B. abortus A19; lane 4 – B. melitensis M5; lane 5 – B. melitensis 16M; and lane 6 – B. ovis; D – agarose gel electrophoresis of species other than Brucella amplified by the conventional PCR, comprising M – DL2000 DNA Marker; lane 1 – B. suis S2; lane 2 – E. coli; lane 3 – S. typhimurium; lane 4 – Y. enterocolitica; lane 5 – V. parahaemolyticus; and lane 6 – RNase-free water

Fig. 6

Analysis of different ratios of viable to dead B. suis S2 strain using the PMA-qPCR method and the qPCR method. * – P < 0.05
Analysis of different ratios of viable to dead B. suis S2 strain using the PMA-qPCR method and the qPCR method. * – P < 0.05

Fig. 7

Determination of the amount of the viable B. suis S2 strain by PMA-qPCR and plate counting methods. Pure bacterial liquid – B. suis cultured in TSB; A – B. suis harbouring in mouse macrophage RAW 264.7 cells infected with 1.28 × 1012 CFU/mL of B. suis S2 strain; B – B. suis harbouring in mouse macrophage RAW 264.7 cells infected with 0.64 × 1012 CFU/mL of B. suis S2 strain; * P < 0.05
Determination of the amount of the viable B. suis S2 strain by PMA-qPCR and plate counting methods. Pure bacterial liquid – B. suis cultured in TSB; A – B. suis harbouring in mouse macrophage RAW 264.7 cells infected with 1.28 × 1012 CFU/mL of B. suis S2 strain; B – B. suis harbouring in mouse macrophage RAW 264.7 cells infected with 0.64 × 1012 CFU/mL of B. suis S2 strain; * P < 0.05

Coefficient of variation for intra- and inter-batch experiments

DNA dilution timesIntra-assayInter-assay
Average value of CTSDCVAverage value of CTSDCV
10717.552770.132050.7517.952030.268621.50
10621.91370.269651.2322.351230.456142.04
10523.806970.150850.6323.828570.436071.83
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Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
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