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

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

doi:10.2478/jvetres-2020-0033

Abstract

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.

References

Arenas-Gamboa A.M., Ficht T.A., Kahl-McDonagh M.M., Gomez G., Rice-Ficht A.C.: The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle. Infect Immun 2009, 77, 877–884.
Search in Google Scholar Back to article
Baily G.G., Krahn J.B., Drasar B.S., Stoker N.G.: Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 1992, 95, 271–275.
Search in Google Scholar Back to article
Bounaadja L., Albert D., Chenais B., Henault S., Zygmunt M.S., Poliak S., Garin-Bastuji B.: Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol 2009, 137, 156–164.
Search in Google Scholar Back to article
Chang C.W., Lin M.H.: Optimization of PMA-qPCR for Staphylococcus aureus and determination of viable bacteria in indoor air. Indoor Air 2018, 28, 64–72.
Search in Google Scholar Back to article
Chen J., Zhao Z., Chen Y., Zhang J., Cao W.: Development and application of a SYBR green real-time PCR for detection of the emerging avian leukosis virus subgroup K. Poult Sci 2018, 97, 2568–2574.
Search in Google Scholar Back to article
Dos Santos L.S., Sa J.C., Dos Santos Ribeiro D.L., Chaves N.P., Da Silva Mol J.P., Santos R.L., Da Paixao T.A., De Carvalho Neta A.V.: Detection of Brucella spp. infection through serological, microbiological, and molecular methods applied to buffaloes in Maranhao State, Brazil. Trop Anim Health Prod 2017, 49, 675–679.
Search in Google Scholar Back to article
Fittipaldi M., Nocker A., Codony F.: Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. J Microbiol Methods 2012, 91, 276–289.
Search in Google Scholar Back to article
Imaoka K., Kimura M., Suzuki M., Kamiyama T., Yamada A.: Simultaneous detection of the genus Brucella by combinatorial PCR. Jpn J Infect Dis 2007, 60, 137–139.
Search in Google Scholar Back to article
Kibbee R.J., Ormeci B.: Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent. J Microbiol Methods 2017, 132, 139–147.
Search in Google Scholar Back to article
Liang N., Dong J., Luo L., Li Y.: Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR. J Food Sci 2011, 76, M234–M237.
Search in Google Scholar Back to article
Liu N.N., Liu Z.S., Hu P., Zhang Y., Lu S.Y., Li Y.S., Yang Y.J., Zhang D.S., Zhou Y., Ren H.L.: Full-Length cDNA Cloning, Molecular Characterization and Differential Expression Analysis of Lysophospholipase I from Ovis aries. Int J Mol Sci 2016, 17, 1206.
Search in Google Scholar Back to article
Liu N.N., Liu Z.S., Lu S.Y., Hu P., Li Y.S., Feng X.L., Zhang S.Y., Wang N., Meng Q.F., Yang Y.J.: Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries. Vet Immunol Immunopathol 2015, 164, 208–219.
Search in Google Scholar Back to article
Liu N.N., Liu Z.S., Lu S.Y., Hu P., Zhang Y., Fu B.Q., Li Y.S., Zhou Y., Zhang Y., Ren H.L.: Isolation and characterisation of peroxiredoxin 6 promoter from sheep Ovis aries J Vet Res 2016, 60, 315–321.
Search in Google Scholar Back to article
Liu Y., Wang C., Tyrrell G., Li X-F.: Production of Shiga-like toxins in viable but nonculturable Escherichia coli O157:H7. Water Res 2010, 44, 711–718.
Search in Google Scholar Back to article
Martirosyan A., Moreno E., Gorvel J.P.: An evolutionary strategy for a stealthy intracellular Brucella pathogen. Immunol Rev 2015, 240, 211–234.
Search in Google Scholar Back to article
Masters C.I., Shallcross J.A., Mackey B.M.: Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction. J Appl Microbiol 2010, 77, 73–79.
Search in Google Scholar Back to article
Mayfield J.E., Bricker B.J., Godfrey H., Crosby R.M., Knight D.J., Halling S.M., Balinsky D., Tabatabai L.B.: The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene 1988, 63, 1–9.
Search in Google Scholar Back to article
Nocker A., Camper A.K.: Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett 2009, 291, 137–142.
Search in Google Scholar Back to article
Nocker A., Richter-Heitmann T., Montijn R., Schuren F., Kort R.: Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing. Int Microbiol 2010, 13, 59–65.
Search in Google Scholar Back to article
Nogva H.K., Drømtorp S.M., Nissen H., Rudi K.: Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. BioTechniques 2003, 34, 804-813.
Search in Google Scholar Back to article
Queipo-Ortuno M.I., Colmenero J.D., Reguera J.M., Garcia-Ordonez M.A., Pachon M.E., Gonzalez M., Morata P.: Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clin Microbiol Infect 2005, 11, 713–718.
Search in Google Scholar Back to article
Sabrina R., Mossadak H.T., Bakir M., Asma M., Khaoula B.: Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria. Vet World 2018, 11, 363–367.
Search in Google Scholar Back to article
Sanjuan-Jimenez R., Colmenero J.D., Bermudez P., Alonso A., Morata P.: Amplicon DNA melting analysis for the simultaneous detection of Brucella spp. and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis. PLoS One 2013, 8, e58353.
Search in Google Scholar Back to article
Soejima T., Iida K., Qin T., Taniai H., Seki M., Yoshida S.: Method to detect only live bacteria during PCR Amplification. J Clin Microbiol 2008, 46, 2305–2313.
Search in Google Scholar Back to article
Wang L.L., Chen X.F., Hu P., Lu S.Y., Fu B.Q., Li Y.S., Zhai F.F., Ju D.D., Zhang S.J., Shui Y.M., Chang J., Ma X.L., Su B., Zhou Y., Liu Z.S., Ren H.L.: Host Prdx6 contributing to the intracellular survival of Brucella suis S2 strain. BMC Vet Res 2019, 15, 304.
Search in Google Scholar Back to article
Wang L.L., Lu S.Y., Hu P., Fu B.Q., Li Y.S., Zhai F.F., Ju D.D., Zhang S.J., Su B., Zhou Y., Liu Z.S., Ren H.L.: Construction and activity analyses of single functional mouse peroxiredoxin 6 (Prdx6). J Vet Res 2019, 63, 99–105.
Search in Google Scholar Back to article
Yan M., Xu L., Jiang H., Zhou Z., Zhou S., Zhang L.: PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state. Microb Pathog 2017, 105, 245–250.
Search in Google Scholar Back to article
Yang Y.J., Liu Z.S., Lu S.Y., Hu P., Li C., Ahmad W., Li Y.S., Xu Y.M., Tang F., Zhou Y., Ren H.L.: Cloning and differential expression analyses of Cdc42 from sheep. J Vet Res 2018, 62, 113–119.
Search in Google Scholar Back to article
Yang Y.J., Liu Z.S., Lu S.Y., Li C., Hu P., Li Y.S., Liu N.N., Tang F., Xu Y.M., Zhang J.H.: Molecular cloning, expression and characterization of programmed cell death 10 from sheep Ovis aries Gene 2015, 558, 65–74.
Search in Google Scholar Back to article
Zhong Q., Tian J., Wang B., Wang L.: PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state. Food Control 2016, 63, 230–238.
Search in Google Scholar Back to article