Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

Mette Sofie Rousing Søndergaard, Charlotta Löfström, Zahra Fares Sayer Al-Habib, M.T. Nielsen, Jeffrey Hoorfar, Mathilde Hasseldam Josefsen

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Abstract

Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity and robustness of a PCR assay, as some polymerases are more resistant to inhibitors, and thus be a simple strategy for assay optimization. Identifying an optimal polymerase can even render costly and time-consuming sample preparation unnecessary.
The aim of this study was to evaluate the performance of 16 commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied on sample matrices known to contain PCR inhibitors (i.e. minced meat samples for Salmonella and chicken fecal samples for Campylobacter).
The samples were prepared for PCR by three methods: No DNA extraction, lysis by boiling and semi-automated DNA extraction for Salmonella and lysis by boiling and two different DNA extraction methods for Campylobacter.
Results show that VeriQuest qPCR master mix have the best general performance, while the AmpliTaq Gold and HotMasterTaq DNA polymerases performed well with meat samples and poorly with fecal samples. Tth DNA polymerase performed well only with the purest DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples.
In conclusion, our data shows that exchanging the DNA polymerase from e.g. Tth to VeriQuest qPCR Master Mix could allow for a less thorough pre-PCR sample preparation with maintained sensitivity, resulting in savings in time and costs.
Original languageEnglish
Publication date2014
Number of pages1
Publication statusPublished - 2014
Event2nd qPCR and Digital PCR Congress - London, United Kingdom
Duration: 20 Oct 201421 Oct 2014
Conference number: 2

Conference

Conference2nd qPCR and Digital PCR Congress
Number2
CountryUnited Kingdom
CityLondon
Period20/10/201421/10/2014

Cite this

Søndergaard, M. S. R., Löfström, C., Al-Habib, Z. F. S., Nielsen, M. T., Hoorfar, J., & Josefsen, M. H. (2014). Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR. Abstract from 2nd qPCR and Digital PCR Congress, London, United Kingdom.
Søndergaard, Mette Sofie Rousing ; Löfström, Charlotta ; Al-Habib, Zahra Fares Sayer ; Nielsen, M.T. ; Hoorfar, Jeffrey ; Josefsen, Mathilde Hasseldam. / Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR. Abstract from 2nd qPCR and Digital PCR Congress, London, United Kingdom.1 p.
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abstract = "Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity and robustness of a PCR assay, as some polymerases are more resistant to inhibitors, and thus be a simple strategy for assay optimization. Identifying an optimal polymerase can even render costly and time-consuming sample preparation unnecessary. The aim of this study was to evaluate the performance of 16 commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied on sample matrices known to contain PCR inhibitors (i.e. minced meat samples for Salmonella and chicken fecal samples for Campylobacter).The samples were prepared for PCR by three methods: No DNA extraction, lysis by boiling and semi-automated DNA extraction for Salmonella and lysis by boiling and two different DNA extraction methods for Campylobacter. Results show that VeriQuest qPCR master mix have the best general performance, while the AmpliTaq Gold and HotMasterTaq DNA polymerases performed well with meat samples and poorly with fecal samples. Tth DNA polymerase performed well only with the purest DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples.In conclusion, our data shows that exchanging the DNA polymerase from e.g. Tth to VeriQuest qPCR Master Mix could allow for a less thorough pre-PCR sample preparation with maintained sensitivity, resulting in savings in time and costs.",
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Søndergaard, MSR, Löfström, C, Al-Habib, ZFS, Nielsen, MT, Hoorfar, J & Josefsen, MH 2014, 'Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR' 2nd qPCR and Digital PCR Congress, London, United Kingdom, 20/10/2014 - 21/10/2014, .

Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR. / Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer; Nielsen, M.T.; Hoorfar, Jeffrey; Josefsen, Mathilde Hasseldam.

2014. Abstract from 2nd qPCR and Digital PCR Congress, London, United Kingdom.

Research output: Contribution to conferenceConference abstract for conferenceResearchpeer-review

TY - ABST

T1 - Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

AU - Søndergaard, Mette Sofie Rousing

AU - Löfström, Charlotta

AU - Al-Habib, Zahra Fares Sayer

AU - Nielsen, M.T.

AU - Hoorfar, Jeffrey

AU - Josefsen, Mathilde Hasseldam

PY - 2014

Y1 - 2014

N2 - Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity and robustness of a PCR assay, as some polymerases are more resistant to inhibitors, and thus be a simple strategy for assay optimization. Identifying an optimal polymerase can even render costly and time-consuming sample preparation unnecessary. The aim of this study was to evaluate the performance of 16 commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied on sample matrices known to contain PCR inhibitors (i.e. minced meat samples for Salmonella and chicken fecal samples for Campylobacter).The samples were prepared for PCR by three methods: No DNA extraction, lysis by boiling and semi-automated DNA extraction for Salmonella and lysis by boiling and two different DNA extraction methods for Campylobacter. Results show that VeriQuest qPCR master mix have the best general performance, while the AmpliTaq Gold and HotMasterTaq DNA polymerases performed well with meat samples and poorly with fecal samples. Tth DNA polymerase performed well only with the purest DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples.In conclusion, our data shows that exchanging the DNA polymerase from e.g. Tth to VeriQuest qPCR Master Mix could allow for a less thorough pre-PCR sample preparation with maintained sensitivity, resulting in savings in time and costs.

AB - Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity and robustness of a PCR assay, as some polymerases are more resistant to inhibitors, and thus be a simple strategy for assay optimization. Identifying an optimal polymerase can even render costly and time-consuming sample preparation unnecessary. The aim of this study was to evaluate the performance of 16 commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied on sample matrices known to contain PCR inhibitors (i.e. minced meat samples for Salmonella and chicken fecal samples for Campylobacter).The samples were prepared for PCR by three methods: No DNA extraction, lysis by boiling and semi-automated DNA extraction for Salmonella and lysis by boiling and two different DNA extraction methods for Campylobacter. Results show that VeriQuest qPCR master mix have the best general performance, while the AmpliTaq Gold and HotMasterTaq DNA polymerases performed well with meat samples and poorly with fecal samples. Tth DNA polymerase performed well only with the purest DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples.In conclusion, our data shows that exchanging the DNA polymerase from e.g. Tth to VeriQuest qPCR Master Mix could allow for a less thorough pre-PCR sample preparation with maintained sensitivity, resulting in savings in time and costs.

M3 - Conference abstract for conference

ER -

Søndergaard MSR, Löfström C, Al-Habib ZFS, Nielsen MT, Hoorfar J, Josefsen MH. Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR. 2014. Abstract from 2nd qPCR and Digital PCR Congress, London, United Kingdom.