An Optimized Method to Assess Viable Escherichia coli O157:H7 in Agricultural Soil Using Combined Propidium Monoazide Staining and Quantitative PCR

Yulong Fu; Zhe Ye; Yangyang Jia; Jiahui Fan; Muhammad Zaffar Hashmi; Chaofeng Shen

doi:10.3389/fmicb.2020.01809

An Optimized Method to Assess Viable Escherichia coli O157:H7 in Agricultural Soil Using Combined Propidium Monoazide Staining and Quantitative PCR

Front Microbiol. 2020 Jul 31:11:1809. doi: 10.3389/fmicb.2020.01809. eCollection 2020.

Authors

Yulong Fu¹, Zhe Ye¹, Yangyang Jia¹, Jiahui Fan¹, Muhammad Zaffar Hashmi², Chaofeng Shen^{1

3}

Affiliations

¹ Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China.
² Department of Chemistry, COMSATS University Islamabad, Islamabad, Pakistan.
³ Zhejiang Provincial Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou, China.

Abstract

Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable Escherichia coli O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 μM PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 10²colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (10⁷ CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of E. coli O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC E. coli O157:H7 in agricultural soil.

Keywords: E. coli O157:H7; PMA; agricultural soils; qPCR; viable cells.