Journal of Environmental Biology

Rapid propidium monoazide cPCR assay for exclusive quantification

of viable Salmonella spp. cells

N. M. Sunar¹*, D.I. Stewart², L.A. Fletcher², E.I. Stentiford² and Muhammad Aqeel Ashraf^3,4

 ¹Department of Civil Engineering Technology, Faculty of Engineering Technology, Universiti Tun Hussein Onn Malaysia, 86400, Batu Pahat,? Johor, Malaysia

²Pathogen Control Engineering (PaCE) Institute, School of Civil Engineering, University of Leeds, Leeds, LS2 9JT, United Kingdom.

³Faculty of Science & Natural Resources, University Malaysia Sabah 88400 Kota Kinabalu, Sabah, Malaysia

⁴Department of Environmental Science and Engineering, School of Environmental Studies, China University of Geosciences, 430074 Wuhan, P. R. China

*Corresponding Author E-mail: shuhaila@uthm.edu.my

 Publication Data

Paper received:

21 February 2016

Revised received:

27 April 2016

Re-revised received:

5 May 2016

Accepted:

23 June 2016

Abstract

Combination of pretreatment with propidium monoazide by competitive polymerase chain reaction (cPCR) was evaluated to enumerate the viability of Salmonella spp. The results showed that PMA treatment was effective in preventing the cPCR detection of target sequences from non-viable cells. In less than 5 hrs, this method generated a signal from viable but nonculturable (VBNC) Salmonella spp. The standard culture method gave approximately 1-2 log¹⁰ cfu ml^-1 less as compared to the PMA-cPCR results. These results provided evidence to support the VBNC state, whereas, the viable cells failed to be cultured by SCM. The proposed method did not detect DNA from dead Salmonella spp. but recognizes the infectious potential of the VBNC state and is thereby, able to assess the effect of control strategies and provide trustworthy data for risk assessment.   

 Key words

Competitive PCR, Pathogen indicator, Propidium monoazide, Salmonella spp.

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