Supplementary Material

Humic substances cause fluorescence inhibition in real-time PCR

Maja Sidstedt1,2, Linda Jansson1, Elin Nilsson3, Laila Noppa3, Mats Forsman3,Peter Rådström1, Johannes Hedman1,2*

1 Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
2 National Forensic Centre, SE-581 94 Linköping, Sweden
3 CBRN Defence and Security, Swedish Defence Research Agency, SE-901 82 Umeå, Sweden

* To whom correspondence should be addressed.
Tel: +46 222 83 29; Email:

Content / Surface water
TOC / 22 mg/L
pH / 6.9
Total N / 930 µg/L
NO2+NO3 / 53 µg/L
Total P / 52 µg/L

Table S1. Characterization of the pool of surface water at the sites where the sediment HS was collected. TOC – total organic carbon, N – nitrogen, NO2 – nitrite, NO3 – nitrate and P - phosphorous.

Table S2.Results of the screening of thefour best performing DNA polymerases. Sediment HS was added to qPCR reactions in different amounts. Results are presented as ∆Cq values relative to the positive controls (PC)(duplicate analysis), i.e. ∆Cqsample=Cqsample-Mean Cqpc. To all reactions, buffer as supplied with each polymerase was used and 16 µg of BSA wasadded asfacilitator.The qPCR was performed as described in Material and methods, except thatprimers (iQFt1F and iQFt1R) and probewere applied as published by Thelaus et al. (Thelaus, Andersson et al. 2009).Genomic DNA (0.4 pg/reaction) from Francisellatularensis was used.ND – Not detected.

Polymerase / ∆Cq for 1 µL sediment HS / ∆Cq for 3 µL sediment HS
Immolase(Bioline) / -0.65;-0.65 / 0.55;0.65
KAPA3G (KAPA Biosystems) / 4.05;3.05 / ND;ND
PerfeCTa Toughmix(Quanta Biosciences) / -1.45;-2.15 / ND;ND
TEMPase(Ampliqon) / -0.45;-0.45 / ND;ND

Table S3.Quenching effect of environmental samples. Performance of the EvaGreen qPCR as described in Material and methods with addition of environmental samples. The effect of landfill soil, plant soil and surface water on the EvaGreen fluorescence was investigated. The end-point fluorescence intensity is shown relative to the positive control without where water was added instead.Strong fluorescence quenching was seen for landfill soil, whereas plant soil caused quenching to a smaller degree. For surface water, no clear fluorescence quenching was noted. NA – Not analysed

Sample / Volume in reaction (µl)
1 / 2 / 4 / 8
Landfill soil / 0.026 / 0.0026 / 0.0013 / NA
Plant soil / 1.5 / 1.2 / 0.74 / 0.54
Surface water / NA / NA / 0.81 / 0.70

Table S4.Humic acid effect on three different DNA polymerases. Results are presented as Cq values (duplicates).Immolase was used as described in Materials and methods. Tempase DNA polymerase (Ampliqon, Odense, Denmark) was used with 10X Tempase ammonium buffer (Ampliqon, 15 mM MgCl2). ExTaq Hot Start DNA polymerase (TaKaRa Bio Inc., Shiga, Japan) was used with 10X ExTaq buffer (TaKaRa Bio Inc., 20 mM MgCl2). Addition of MgCl2 to the buffer was made to obtain a final concentration of 4 mM. Hydrolysis probe was used as described in Materials and methods. Either 1 U or 2 U DNA polymerase was added to each reaction. ND – Not detected

Humic acid
amount (ng) / Immolase, 1U / Immolase, 2U / Tempase, 1U / Tempase, 2U / ExTaqHS, 1U / ExTaqHS, 2U
0 / 29.3;29.2 / 29.4;29.4 / 29.7;29.8 / 29.8;29.8 / 28.5;28.4 / 28.9;28.9
20 / 29.4;29.4 / 29.4;29.4 / 30.0;29.9 / 29.8;29.8 / 28.3;28.3 / 28.7;28.7
100 / 29.5;29.5 / 29.4;29.4 / 31.5;31.7 / 30.2;30.2 / 27.8;27.8 / 28.4;28.4
300 / 29.5;29.5 / 29.5;29.5 / 41.6;43.4 / 36.0;37.1 / 37.2;37.7 / 28.2;28.2
500 / 29.7;29.6 / 29.5;29.4 / ND;ND / 46.9;47.4 / ND;ND / 33.4;33.8
900 / 37.4;38.3 / 30.3;30.6 / ND;ND / ND;ND / ND;ND / ND;ND
1000 / 38.6;38.6 / 33.5;32.7 / ND;ND / ND;ND / ND;ND / ND;ND

Table S5.Effect of BSA on HA amplification inhibition. Addition of different amountsof HA and BSA tohydrolysis probe qPCR reactions with Immolase. Results are presented as Cq values (duplicates). NA – Not analysed, ND – Not detected.

BSA amount(µg)
Humic acid amount (ng) / 0 / 0.25 / 0.5 / 1 / 2 / 5 / 10 / 15 / 20
0 / 29.5;29.4 / 29.3;29.2 / 29.2;29.3 / 29.2;29.3 / 29.2;29.3 / 29.4;29.5 / 29.3;29.4 / 29.4;29.4 / 29.3;29.4
20 / 29.6;29.6 / 29.1;29.2 / 29.1;29.2 / 29.3;29.4 / 29.3;29.3 / 29.4;29.5 / 29.4;29.4 / NA / NA
100 / ND;ND / 34.8;35.0 / 30.2;31.2 / 29.4;29.5 / 29.3;29.4 / 29.5;29.5 / 29.5;29.5 / NA / NA
300 / ND;ND / ND;ND / ND;ND / ND;ND / 43.7;43.9 / 29.8;29.5 / 29.4;29.5 / NA / NA
500 / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / 34.8;34.5 / 29.5;29.6 / 29.4;29.5 / 29.4;29.4
1000 / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / 51.3;47.3 / 35.0;35.2 / 31.0;31.6
1500 / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / 49.8;ND / 45.1;ND
2000 / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND / ND;ND

Figure S1.Emission spectra for the EG-DNA complex in presence and absence of humic acid (HA). The fluorescence intensity is plotted as intensity relative to the maximum fluorescence value for the positive control, 50 ng HA and 100 ng HA, respectively. Addition of HA does not cause a shift in the fluorescence spectra.

References

Thelaus, J., A. Andersson, P. Mathisen, A. L. Forslund, L. Noppa and M. Forsman (2009). Influence of nutrient status and grazing pressure on the fate of Francisella tularensis in lake water.FEMS Microbiol Ecol67(1): 69-80.