Molecular diagnostics of <i>Salmonella</i> and <i>Campylobacter</i> in human/animal fecal samples remain feasible after long-term sample storage without specific requirements

CB Harder; S Persson; J Christensen; A Ljubic; EM Nielsen; J Hoorfar; CB Harder; S Persson; J Christensen; A Ljubic; EM Nielsen; J Hoorfar

doi:10.3934/microbiol.2021024

AIMS Microbiology

2021, Volume 7, Issue 4: 399-414. doi: 10.3934/microbiol.2021024

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Research article

Molecular diagnostics of Salmonella and Campylobacter in human/animal fecal samples remain feasible after long-term sample storage without specific requirements

1.
Statens Serum institut, Dept. Bacteriology, Parasitology and Fungi, Artillerivej 5, 2300 Copenhagen, Denmark
2.
Molecular Ecology, Microbial Ecology and Evolutionary Genetics, Lund University, Sölvegatan 37, 223 62 Lund
3.
Danish Veterinary and Food Administration, Microbiological department, Søndervang 4, 4100 Ringsted
4.
AGC Biologics, Process Transfer, Vandtårnsvej 83, 2860 Søborg, Denmark
5.
Technical University of Denmark, National Food Institute, 2800 Kgs. Lyngby, Denmark

Received: 17 March 2021 Accepted: 05 September 2021 Published: 14 October 2021

Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives.

This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 10⁶ CFU/mL of both Salmonella and Campylobacter strains stored at −20 °C, 5 °C and 20 °C (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.
- Detection threshold,
- Campylobacter,
- Salmonella,
- gastritis,
- DNA stability,
- diagnostics,
- preservation methods,
- pathogens
Citation: CB Harder, S Persson, J Christensen, A Ljubic, EM Nielsen, J Hoorfar. Molecular diagnostics of Salmonella and Campylobacter in human/animal fecal samples remain feasible after long-term sample storage without specific requirements[J]. AIMS Microbiology, 2021, 7(4): 399-414. doi: 10.3934/microbiol.2021024

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Abstract

Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives.

This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 10⁶ CFU/mL of both Salmonella and Campylobacter strains stored at −20 °C, 5 °C and 20 °C (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.

Acknowledgments

This research was supported by InnovationsFonden (formerly called The Danish National Advanced Technology Foundation) to the project ‘Metagenome Kit’ (Grant number: 85-2013-1). CBH was supported by the Carlsberg Foundation (grants no. CF18-0809/CF19-0197) at the time of writing. This publication reflects the views only of the author/(s) and neither InnovationsFonden nor the Carlsberg Foundation can be held responsible for any use which may be made of the information contained therein. We would like to thank Christian Vråby Pedersen for excellent technical assistance.

Conflict of interest

The authors declare no conflict of interest.

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