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Updated: Jul 16, 2026
Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples
Published on: October 25, 2018
Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR
Carsten Suhr Jacobsen1, William E Holben
1Geological Survey of Denmark and Greenland, DK-1350, Copenhagen, Denmark. csj@geus.dk
Abstract:
Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR.
Insights
Quantifying Salmonella mRNA in soil and manure is now possible using magnetic capture hybridization. This method accurately detects Salmonella sp. in agricultural soil, outperforming DNA-based detection by minimizing false positives.
Area of Science:
- Environmental microbiology
- Molecular biology
- Food safety
Background:
- Accurate detection of Salmonella sp. in environmental samples like soil and manure is crucial for public health and food safety.
- Traditional methods for Salmonella detection can be time-consuming and may yield false positives, especially in complex matrices.
- Quantifying viable bacterial populations requires methods that target actively expressed genes, such as mRNA.
Purpose of the Study:
- To develop and validate a method for the direct quantification of Salmonella sp. mRNA in soil and chicken manure.
- To assess the efficacy of magnetic capture hybridization coupled with RT-qPCR for Salmonella mRNA detection.
- To compare the performance of mRNA-based detection with DNA-based detection in environmental samples, particularly after pasteurization.
Main Methods:
- Direct quantification of Salmonella sp. mRNA using magnetic capture hybridization and reverse transcription quantitative PCR (RT-qPCR).
- Targeting the invA gene specific to Salmonella sp. for detection.
- Purification of RNA using phenol/chloroform extraction, isopropanol precipitation, and DNase treatment to remove contaminating DNA.
Main Results:
- The developed method successfully quantified Salmonella sp. mRNA in high organic agricultural soil with a detection limit of 5 x 10^4 cells per gram.
- The presence of chicken manure (1:4 w/w) did not impede the quantification of Salmonella sp. mRNA.
- Pasteurization of chicken manure significantly reduced invA mRNA detection, whereas DNA-based qPCR still detected Salmonella, indicating mRNA detection's specificity to viable cells.
Conclusions:
- Magnetic capture hybridization combined with RT-qPCR provides a robust and sensitive method for direct mRNA quantification of Salmonella sp. in soil and manure.
- mRNA-based detection is superior to DNA-based qPCR for minimizing false-positive results, especially in samples treated to inactivate bacteria.
- This technique offers a valuable tool for assessing the presence and viability of Salmonella in agricultural environments, contributing to improved food safety monitoring.
