Al-Habib, Zahra Fares Sayer

Hoorfar, Jeffrey

Josefsen, Mathilde Hasseldam

Löfström, Charlotta

Nielsen, M.T.

Søndergaard, Mette Sofie Rousing

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 20, 2017Polymerase study: Improved detection of Salmonella and Campylobacter through theoptimized use of DNA polymerases in diagnostic real-time PCRFachmann, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer; Nielsen, M.T.;Hoorfar, Jeffrey; Josefsen, Mathilde HasseldamPublication date:2014Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):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 DNApolymerases in diagnostic real-time PCR. Poster session presented at 2nd qPCR and Digital PCR Congress,London, United Kingdom.Screening: Table 1. 16 DNA polymerases and 4 master mixes screened in standardized Salmonella and Campylobacter qPCR assays2,3 on a 10-fold dilution series of purified DNA, ranked after performance. Examples of good, intermediate, and poor performance on high to low DNA purity: Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic  real-time PCR Aim To investigate the performance of different DNA polymerases and master mixes in real-time PCR and their resistance towards inhibitors in  matrices relevant for food safety, using validated PCR assays for Salmonella and Campylobacter. Mette S.R. Søndergaard, C. Löfström, Z.F.S. Al-Habib, M.T. Nielsen, J. Hoorfar and M.H. Josefsen  National Food Institute, Technical University of Denmark.          Conclusions • The performances of the tested DNA polymerases varied considerably, reinforcing the importance of careful selection of an appropriate DNA polymerase for the PCR assay and sample type in question. • For Salmonella in minced meat samples, HotMaster Taq, AmpliTaq Gold and VeriQuest were found to be the best performing alternative DNA polymerases.  • For Campylobacter in chicken feces samples, VeriQuest and ExTaq were found to be the best performing alternative DNA polymerases.      Table 2. The performance of the four polymerases and the master mix, with the best results in the screening, on three different DNA extractions methods on: A. Meat artificially contaminated with Salmonella and  B. Feces artificially contaminated with Campylobacter. Materials and Methods 16 commercially available DNA polymerases and 4 master mixes were included (see Table 1) These were evaluated on a dilution series of purified Salmonella ser. Typhimurium and Campylobacter jejuni DNA, analyzed by standardized real-time PCR assays2,3 using the accompanying PCR buffers for each polymerase.  The 5 best performing polymerases/kits were further evaluated using minced pork meat samples (diluted in BPW 1:10 and enriched for 18 h at 37°C followed by artificial contamination with Salmonella ser. Typhimurium ,102-106 CFU/ml) and chicken feces samples (artificially contaminated with Campylobacter jejuni, 102-106 CFU/ml). DNA extraction was performed on the samples by three different methods (Magnetic beads-based (KingFisher), lysis by boiling, and non-extracted for Salmonella and QIAamp® Fast DNA Stool Mini Kit (Qiagen), magnetic beads-based, and lysis by boiling for Campylobacter) followed by real-time PCR. Polymerases were rated based on shape of amplification curves, amplification efficiency (AE), linear range and linearity of standard curve (R2) and max flourescence (Max dR)   References 1. Hedman et al (2012) Pre-PCR processing strategies. In: PCR Technologies, current innovations CRC Press, 3rd Ed. 2. Löfström et al  (2009) BMC Microbiol, 9(1):85.  3. Josefsen et al (2010) AEM, 76(15):5097.  Acknowledgements Julia Christensten, Kirsten Michaëlis, and Pia Engelsmann are acknowledged for providing excellent technical assistance. Financial support by the Danish Council for Strategic Research project CamVac (contract 09-067131) and the Ministry of Food, Agriculture and Fisheries of Denmark, the GUDP project UltraSal (grant no. 3405-11-0349). A.  DNA Polymerase or master mix Magnetic beads based DNA extraction Lysis by boiling Non-extracted Rating LoD [CFU/ml] Max dR Rating LoD [CFU/ml] Max dR Rating LoD [CFU/ml] Max dR Tth ++ 102* 59615 + 103 47634 + 105 11710 VeriQuest MM  ++ 102* 28736 ++ 102 17216 ++ 104 7430 AmpliTaq Gold ++ 102* 64637 ++ 102* 147690 ++ 104 12874 HotMaster Taq ++ 102 35691 +++ 102* 97583 ++ 104 23048 TaKaRa ExTaq HS - 103 27058 - 104 47569 - ND 257 B.  DNA Polymerase or master mix Qiagen kit extraction Magnetic beads based DNA extraction Lysis by boiling Rating LoD [CFU/ml] Max dR Rating LoD [CFU/ml] Max dR Rating LoD [CFU/ml] Max dR Tth ++ 103 22022 - NA - 106 SD VeriQuest MM  ++ 102 12661 - NA + 104 5578 AmpliTaq Gold + 103 6643 - NA - NA HotMaster Taq + 103 9088 - NA - 106 SD TaKaRa ExTaq HS ++ 102 12661 + 103 7861 - 106 SD +++ Very good, ++ Good, + Intermediate, - Poor, NA no amplification, SD single detection, *LoD could be lower, but only 102 to 106 CFU/ml was tested DNA Polymerase or Master mix Performance on Salmonella Performance on Campylobacter Con-clusion  Price [USD/U] Rating LoD [µg/ml] Max dR Rating LoD [µg/ml] Max dR Tth DNA Polymerase  (Roche) +++ 1.2 × 10-6 42602 +++ 6.6 × 10-7 48971 +++ 1.22 VeriQuest™ Probe qPCR Master Mix (Affymetrix) +++ 1.2 × 10-5* 38063 +++ 6.6 × 10-7 43067 +++ 0.92 AmpliTaq Gold®  (Applied Biosystems) +++ 1.2 × 10-6 34546 ++ 6.6 × 10-7 28147 +++ 0.75 HotMaster® Taq DNA Polymerase  (5 Prime) +++ 1.2 × 10-5* 39244 ++ 6.6 × 10-7 16543 +++ 0.66 TaKaRa Ex Taq® Hot Start Version  (TaKaRa Bio Inc) +++ 1.2 × 10-5* 37222 ++ 6.6 × 10-7 24549 +++ 1.04 AmpliTaq® DNA Polymerase  (Applied Biosystems) ++ 1.2 × 10-5* 40053 ++ 6.6 × 10-7 24930 ++ 0.80 AmpliTaq® 360 DNA Polymerase  (Applied Biosystems) ++ 1.2 × 10-5* 39484 ++ 6.6 × 10-7 35547 ++ 0.56 AmpliTaq Gold® 360 DNA Polymerase  (Applied Biosystems) +++ 1.2 × 10-5 41110 ++ 6.6 × 10-7 28090 ++ 1.29 TaqMan® Fast Advanced Master Mix  (Applied Biosystems) ++ 1.2 × 10-6 34546 ++ 6.6 × 10-6 28331 ++ 1.20 SG qPCR Master Mix  (EURx) +++ 1.2 × 10-6 30894 + 6.6 × 10-6 6996 IC 0.46 HotStarTaq® Master Mix kit  (Quiagen) +++ 1.2 × 10-5* 37751 + 6.6 × 10-4 14648 IC 0.99 PicoMaxx High Fidelity PCR System  (Agilent Technologies) + 1.2 × 10-3 24797 ++ 6.6 × 10-6 19700 IC 1.00 FastStart Taq DNA Polymerase  (Roche) +++ 1.2 × 10-5* 39644 - 6.6 × 10-2 5091 IC 1.07 MyTaq™ HS DNA polymerase  (Bioline) + 1.2 × 10-5 5448 + 6.6 × 10-7 5338 + 0.66 MyTaq™ DNA Polymerase  (Bioline) + 1.2 × 10-5 5126 + 6.6 × 10-6 5733 + 0.33 Titanium™ Taq DNA Polymerase  (Clontech) - 1.2 × 10-5 688 + (72°C) 6.6 × 10-6 8584 - 2.68 OneTaq® DNA Polymerase  (New England BioLabs) - ND + 6.6 × 10-5 12860 - 0.20 Phusion® High-Fidelity DNA Polymerase with GC buffer (New England BioLabs) - 1.2 × 10-5 2676 - ND - 0.84 Pfu DNA Polymerase  (Fermentas) - 1.2 2358 - ND - 0.68 Herculase II Fusion DNA Polymerase  (Agilent Technologies) - ND - ND -  0.07 +++ Very good, ++ Good, + Intermediate, - Poor, ND not detected, IC inconclusive, *LoD could be lower. Further evaluation of top 5: -10.000010.00020.00030.00040.00050.00010 15 20 25 30 35 40Flourescence [dR] Cycle no. Figure 3. Performance on non-extracted meat samples  VeriQuest, 10^5Tth 10^5VeriQuest, 10^3Tth 10^3ExTaq 10^5ExTaq 10^3-10.000010.00020.00030.00040.00050.00010 15 20 25 30 35 40Flourescence [dR] Cycle no. Figure 2. Performance on lysis by boiling extracted meat samples VeriQuest, 10^5Tth 10^5ExTaq 10^5VeriQuest, 10^3Tth 10^3ExTaq 10^3-10.000010.00020.00030.00040.00050.00010 15 20 25 30 35 40Flourescense [dR] Cycle No. Figure 1. Performance on magnetic beads-based extracted meat samples Tth, 10^5VeriQuest, 10^5ExTaq, 10^5Tth, 10^3VeriQuest, 10^3ExTaq, 10^3Mette Sofie Rousing Søndergaard PhD Student National Food Institute, DTU Division of Food Microbiology msrso@food.dtu.dk Introduction To improve food safety it is important to pursue fast, well performing and low-cost methods for detection  of foodborne pathogens. Though real-time PCR offers several advantages compared with classical  microbiology, the choice of a suitable DNA polymerase has been shown to optimize method performance considerably1.    Decreasing purity of DNA extractions Decreasing purity of DNA extractions 

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

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.<br/

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Dec 20, 2017Polymerase study: Improved detection of Salmonella and Campylobacter through theoptimized use of DNA polymerases in diagnostic real-time PCRFachmann, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer; Nielsen, M.T.;Hoorfar, Jeffrey; Josefsen, Mathilde HasseldamPublication date:2014Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):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 DNApolymerases in diagnostic real-time PCR. Abstract from 2nd qPCR and Digital PCR Congress, London, UnitedKingdom.Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR  Mette S. R. Søndergaard, Charlotta Löfström, Zahra F. S. Al-Habib, Mette T. Nielsen, Jeffrey Hoorfar and Mathilde H. Josefsen. National Food Institute, Technical University of Denmark, Søborg, Denmark. Email: msrso@food.dtu.dk  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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Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

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