Development of PMAxxTM-Based qPCR for the Quantification of Viable and Non-viable Load of Salmonella From Poultry Environment.

Jiawei Zhang,Samiullah Khan,Kapil K Chousalkar

doi:10.3389/fmicb.2020.581201

Abstract

Determining the viable and non-viable load of foodborne pathogens in animal production can be useful in reducing the number of human outbreaks. In this study, we optimized a PMAxxTM-based qPCR for quantifying viable and non-viable load of Salmonella from soil collected from free range poultry environment. The optimized nucleic acid extraction method resulted in a significantly higher (P < 0.05) yield and quality of DNA from the pure culture and Salmonella inoculated soil samples. The optimized primer for the amplification of the invA gene fragment showed high target specificity and a minimum detection limit of 102 viable Salmonella from soil samples. To test the optimized PMAxxTM-based qPCR assay, soil obtained from a free range farm was inoculated with Salmonella Enteritidis or Salmonella Typhimurium, incubated at 5, 25, and 37°C over 6 weeks. The survivability of Salmonella Typhimurium was significantly higher than Salmonella Enteritidis. Both the serovars showed moisture level dependent survivability, which was significantly higher at 5°C compared with 25°C and 37°C. The PMAxxTM-based qPCR was more sensitive in quantifying the viable load compared to the culture method used in the study. Data obtained in the current study demonstrated that the optimized PMAxxTM-based qPCR is a suitable assay for quantification of a viable and non-viable load of Salmonella from poultry environment. The developed assay has applicability in poultry diagnostics for determining the load of important Salmonella serovars containing invA.

Highlights

Food safety is an important aspect of human health and, ; state health departments are routinely tasked to trace back foodborne pathogens for their source of origin
109 colony forming unit (CFU)/mL of Salmonella broth culture was centrifuged at 19,500 × g for 3 min and the pellet was resuspended in 100 μL phosphate buffered saline (PBS) into which 300 μL tissue lysis buffer (ATL) and half spoon of acids washed 106 μm beads (Sigma Aldrich, Australia) were added
Primer optimization steps showed that primers for the ompA gene were non-specific and amplified the genomic DNA of Escherichia coli

Summary

Introduction

Food safety is an important aspect of human health and, ; state health departments are routinely tasked to trace back foodborne pathogens for their source of origin. Zoonotic pathogens, such as Salmonella and Campylobacter, often result in human gastroenteritis after the consumption of contaminated poultry products (Kärenlampi et al, 2007; Ford et al, 2018). There has been an increase in free range poultry production over the years. In a free-range poultry production system, birds have access to the range area outside poultry shed. Infected chickens can shed Salmonella to their environment intermittently.

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