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Characterization of the Effects of Heat Stress on the DNA-Intercalating Dye EvaGreen for Potential Use With the Joint Biological Agent Identification and Diagnostic System

June 1, 2014

Veverka, Donald V

ABSTRACT Although advances in real-time polymerase chain reaction (PCR) technology and equipment have facilitated field research, only a limited selection of reagents do not require cold storage. This study explored the temperature stability of the commercially available DNA-intercalating dye EvaGreen after exposure to a spectrum of temperatures for 176 days by analyzing quantification cycle (Cq) and end fluorescence levels during amplification of the invA gene of Salmonella typhimurium. To further characterize potential dye stability, the effects of small differences in dye volume were examined and dye samples were subjected to an Air Force deployment to the Middle East. Significant differences in Cq and end fluorescence were found; however, the magnitude of mean Cq differences was less than one cycle and the magnitude of mean fluorescence differences was less than that attributable to a difference of 0.25 L of dye per 25 L reaction. Liquid EvaGreen dye may thus be stable at temperatures as high as 65 C for up to 6 months for use in real-time PCR. These results warrant further investigation by using liquid EvaGreen dye to adapt traditional lab-based real-time PCR assays for Joint Biological Agent Identification and Diagnostic System use and testing the assays in the field.


The development of real-time polymerase chain reaction (PCR) has provided scientists a rapid and reliable method of target-gene identification and amplification.1,2 Real-time PCR utilizes fluorescent dyes in conjunction with standard PCR master mixes to directly quantify a target amplicon during thermocycling3 and does not require post-PCR detection procedures, such as electrophoresis and visualization, needed for standard PCR.4 This technology has played a direct role in the understanding of gene expression, detection of genetic abnormalities and cancers, surveillance of public health, and diagnosis of human pathogens.5-9

The rapid detection of DNA molecular signatures is an increasingly important, sensitive, and specific method for identifying pathogens and vectors under field conditions. Military personnel can literally carry portable field platforms such as the U.S. Military's Joint Biological Agent Identification and Diagnostic System (JBAIDS) on their backs.10 Assays optimized for JBAIDS use have been shown to be comparable to assays performed in the lab using equipment such as the ABI 7500 Fast instrument, demonstrating the potential to adapt lab assays for JBAIDS use.11 Many of the reagents used in a typical laboratory setting require cold storage, but established infrastructure is rarely available in the developing world and resources for cold storage are often expensive and inadequate.12,13 Therefore, the availability of a stable and customizable reagent master mix that requires only the addition of template and molecular-grade water would facilitate the use of real-time PCR in the field. No currently available commercial product, however, has all of the requisite properties for a thermostable, flexible, cost-effective real-time PCR assay. Fully lyophilized PCR mixes such as illustra PuReTaq Ready-To-Go PCR beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK)14 are shelf-stable for up to 32 months and contain deoxynucleotide triphosphates (dNTPs), Mg2+, buffer, and Taq polymerase but do not contain a real-time PCR dye. Biofire Diagnostics (Salt Lake City, Utah; previously Idaho Technology) offers freeze-dried reagent kits that contain fluorescent probes and are stable at room temperature. However, there is a limited selection of pathogen-specific primer-probe sets accessible to JBAIDS operators, severely limiting the ability to customize molecular assays to meet emergent threats or cost-effectively prepare the reagents needed to detect a range of pathogens. The stability of the fluorescent dyes is an issue,15 as the degradation of both intercalating dyes and TaqMan (Life Technologies, Carlsbad, California) probes limits the ability of long-term field research.16 If an intercalating dye were thermostable at 65 C (a temperature often seen in deployed settings in the Middle East), a technician could use a single dye with multiple primer sets. As it is possible to order primers with custom sequences quickly and inexpensively, this system would greatly increase flexibility and reduce cost.

Traditionally, it has been assumed that lyophilization is necessary for the long-term preservation of real-time PCR reagents without cold storage,17,18 as commercial liquid dyes, such as SYBR Green (Life Technologies, Carlsbad, California) and Qiagen QuantiFast (Qiagen Inc., Valencia, California), have shown degradation after a 1-month exposure to 45 C.15 However, lyophilization may not be the ideal solution, as the commercially available qPCR GreenMaster (Jena Bioscience, Jena, Germany), a lyophilized qPCR master mix containing the DNA-intercalating dye EvaGreen (Biotium Inc., Hayward, California), has a recommended storage temperature of -20 C and is described as stable at 4 C for only 3 months. Preliminary research in our lab (data not shown), however, suggested that EvaGreen dye demonstrates temperature stability without lyophilization.

The purpose of this study was to investigate the thermostability of the real-time PCR dye EvaGreen and to characterize a robust, flexible, and temperature-stable PCR master mix using EvaGreen dye in conjunction with GE Ready-To-Go PCR beads.


DNA Isolation

Samples of Salmonella typhimurium (ATCC 14028) were prepared from fresh, overnight cultures grown in tryptic soy broth. DNA was isolated at a concentration of 11 ng/L according to previously published procedures19 and diluted to a working concentration of 0.5 ng/L.

Real-Time PCR

The Salmonella invasion protein gene invA was chosen as the target amplicon (284 bp).20 The previously published forward primer InvA-Fwd [5'-GTGAAATTATCGCCACGTTCGG GCAA-3'] and reverse primer InvA-Rev [5'-TCATCGCA CCGTCAAAGGAACC-3'] (Integrated DNA Technologies, Coralville, Iowa) were suspended in sterile molecular-grade water at a concentration of 100 pmol/L and diluted to a working concentration of 20 pmol/L.

Real-time PCR and data analysis were performed using the Applied Biosystems 7900HT Fast Real-Time PCR system (Life Technologies, Carlsbad, California). The PCR mixture (24 L) contained one illustra puReTaq Ready-To-Go PCR bead (GE Healthcare), 20 pmol of each primer (InvA-Fwd, InvA-Rev), 1.25 L of EvaGreen dye, and 1.0 ng of the S. typhimurium DNA used as the template, all in molecular-grade water. The reaction bead contains stabilizers, BSA, dNTPs, ~2.5 U of puReTaq DNA polymerase, and reaction buffer.21

Amplification conditions were 90 Cfor5minutesfollowed by 40 cycles of PCR consisting of 10 seconds at 95 C, 15 seconds at 52 C, and 15 seconds at 72C. Performance was evaluated using quantification cycle (Cq) and end fluorescence in fluorescence spectral units (FSU). Cq values were determined at a threshold of 0.70 ? Rt, using a manual baseline of cycles 3-12.

Time Trials

EvaGreen dye at a concentration of "20 in water" from a single lot was portioned into 600 individual samples each containing 15 L of dye in a 1.5 L snap cap microcentrifuge tube. Sets of 150 samples each were stored at 25 C, 45 C, and 65 C, and a control temperature of 4 C protected from light. Time trials were conducted approximately every 2 weeks for 3 months and on a monthly basis for another 3 months. When evaporation occurred under storage conditions, the dyes were reconstituted to 15 L shortly before use in PCR. All runs included a no template control (NTC) with no DNA. In total, 48 samples were tested per trial (11 samples with DNA and 1 NTC for each temperature condition). The position of temperature groups within the 96-well plate was randomized among runs.

Dye Volume Trial

The real-time PCR protocol for the dye volume trials is identical to that described for the time trials except for dye volume. Five dye volumes (0.75, 1.00, 1.25, 1.50, and 1.75 L) were tested. Total reaction volumes were made equal with additional molecular-grade water.

Field Conditions Trial

Before deployment, baseline real-time PCR was run using a previous lot of EvaGreen dye stored at 4 C as described above except for using 0.75 ng of a previous preparation of Salmonella DNA as the template. To prepare samples, 15-L aliquots of dye were placed in screw-top, gasket-type plastic vials, wrapped in aluminum foil, and either taken into the field (Ali Al-Salem Air Force Base, Kuwait) or stored at 4 C. Other than using aluminum foil to protect the dye from sunlight, no other actions were taken to stabilize the dye or to control the dye's temperature throughout the deployment.

During the 34-day deployment, the dye was exposed to temperatures usually ranging from 19 Cto45 C and occasionally rising to 50 C. Upon return to the United States, evaporation of the dyes had occurred to some extent; therefore, all samples were reconstituted to 15 L before testing. Real-time PCR was performed on both the dye exposed to field conditions and the control dye simultaneously.

Data Analysis

Data were analyzed using the Sequence Detection System software version 2.3 using the FAM-BHQ1 detector format. NTCs gave Cq values of 19.5 to 29.2, well above sample Cq values. NTCs yielded fluorescence values of 3,161 to 14,371 FSU but could be readily distinguished from positives by dissociation curve analysis. Statistical analysis was conducted using the JMP 10.0.0 Statistics program (SAS Institute Inc., Cary, North Carolina).


The initial goal of this study was to pursue heat-stress trials of lyophilized samples of commercially available intercalating dyes. However, the complexities involved in lyophilizing light-sensitive dyes and the requirement for specialized lyophilization equipment led to investigation of the thermostability of the dyes in liquid form. To explore the thermostability of liquid EvaGreen dye, a trial was designed to test multiple temperature conditions over time. EvaGreen dye was stored at 25 C, 45 C, and 65 C for 176 days to simulate possible conditions faced by technical personnel in deployed environments, and the real-time PCR performance of heat-stressed dye was compared to that of control dye stored at 4 C.

Time Trial Cq

The mean Cq values (with standard error) for 10 independent samples per temperature per time point are listed in Table I. ANOVA by temperature revealed significant differences between some groups. However, comparison by Student's t-tests (Fig. 1A) showed that despite the harsh conditions throughout the experiment, the higher temperature means, 45 C and 65 C, showed no statistical difference from the 4 C control group. The 25 C group had the highest Cq values, but there was no statistically significant difference between the 25 C and 45 C groups. The mean Cq value of 25 C dye (15.49 0.03 Cq) was within 0.2 Cq values of the 4 C and 65 C means. Although such a difference does have statistical significance, it is not expected this would have an effect on overall experimental outcome for most users.

ANOVA by day revealed significant differences between groups, and comparison by Student's t-tests (Fig. 1B) showed slight but significant differences in mean Cq values throughout the study. The largest difference between two groups was 0.86 Cq. Day 7, a run used to establish a baseline, had the lowest mean Cq value (14.82 0.04 Cq), whereas Day 69 had the highest mean Cq value (15.68 0.03 Cq). There are no detectable patterns in the statistical variability between the days, and the small magnitude of the variation would not be expected to impact the overall outcome of a field-PCR experiment.

Time Trial End Fluorescence

End fluorescence is the detectable fluorescence measured by a real-time PCR instrument. Although fluorescence is a variable closely associated with Cq values, end fluorescence measurements show the absolute and cumulative performance of the dye throughout the PCR run. Because Cq values are derived from the increase in fluorescence over baseline levels (cycles 3 -12) during the remaining cycles, it may not reveal dye degradation if both starting and ending fluorescence are diminished. For this reason, end fluorescence can provide further insight into dye stability and signal robustness. The fluorescence values are measured using FSU determined by the ABI 7900 software.

The mean end fluorescence values (with standard error) for 10 independent samples per temperature per time point are listed in Table II. ANOVA by temperature revealed significant differences between groups, and comparison by Student's t-tests (Fig. 2A) showed that the means of the 4 Cgroup (15,868 253 FSU) and the 45C group (15,351 263 FSU) were statistically different from the means of the 25 Cgroup (14,478 248 FSU) and the 65C group (13,755 285 FSU). Interestingly, both the highest temperature (65 C) and room temperature (25 C) groups showed significantly lower fluorescence levels than the control (4 C) whereas the 45 C group did not. Although it is not surprising that storage at a very high temperature (65 C) may have caused a small amount of dye degradation, the basis for similar degradation at room temperature (25 C) requires further investigation to elucidate.

It should be noted that EvaGreen dye is suspended in molecular-grade water when purchased. Because of the applied heat stress and low humidity in Colorado, where this study was performed, the water component of the 45 C and 65 C dye vaporized into the samples' tubes in 24 to 48 hours, leaving a residue behind. These samples were resuspended in the correct volume of molecular-grade water before performing real-time PCR. The 25 C samples vaporized more slowly, containing liquid for up to 14 days. Great care was taken to return each aliquot to the correct volume before being used for experimentation to prevent the concentration of EvaGreen dye from varying between samples; however, it is possible that some variation did occur because of systematic and random pipette error, especially in cases of incomplete vaporization. The apparent degradation in the 25 C samples compared to the 45 C samples could be related to the difference in water evaporation time and may warrant further investigation.

ANOVA by day revealed significant differences between groups, and comparison by Student's t-tests (Fig. 2B) showed fluorescence variability over time, but all means remained well above 10,000 FSU, which is generally considered a robust signal. The highest mean fluorescence (16,039 555 FSU) was on Day 69 and the lowest mean (14,168 415 FSU) was on Day 28. The results showed no predictable pattern of statistical significance in fluorescence between the days. Although statistically significant differences were present, all mean EvaGreen fluorescence levels were above 10,000 FSU, which should allow for satisfactory real-time PCR performance.

Dye Volume Trial

The need to reconstitute evaporated dye samples by adding molecular-grade water to the dry sample tubes immediately before use introduces a small possibility of using dye at a reaction concentration slightly different from manufacturer recommendations. To explore the possible volume effects of EvaGreen dye, different volumes (and thus different reaction concentrations) of dye were evaluated in the real-time PCR reagent mix. The mean Cq and end fluorescence values (with standard error) for 10 independent samples per dye volume are plotted in Figure 3. ANOVA by dye volume revealed significant differences between groups, and comparison by Student's t-tests (Fig. 3) showed significant differences in both Cq values and mean fluorescence. The manufacturer's recommended dye volume per 25 L reaction (1.25 L) had the lowest Cq value (15.73 0.08 Cq), whereas the 0.75 L samples had the highest overall mean (16.23 0.06 Cq). The lower volumes of dye may not provide enough dye molecules to bind to all the amplified DNA whereas the higher volumes of dye likely caused reaction inhibition, resulting in less DNA amplification and higher Cq values.

Although the highest mean Cq value (16.23 0.06 Cq) was only 3.2% higher than the lowest mean Cq value (15.73 0.08 Cq), the effect of dye volume on fluorescence was much more apparent. There is an approximately linear increase in dye fluorescence as volume is increased from 0.75 Lto 1.5 L. It is presumed that either the fluorescence detector of the 7900HT became saturated or that reaction inhibition prevented continuation of the linear trend up to 1.75 L per reaction. The 1.75 L samples showed higher mean fluorescence (22,258 660 FSU), an increase of 144% over the lowest mean fluorescence, than that of the 0.75 L samples (9,126 574 FSU). The mean of the 1.0 L samples (13,690 523 FSU) is 5,281 FSU lower than the mean of the manufacturer-recommended volume of 1.25 L(18,971 724 FSU). These data suggest that small changes in fluorescence, such as those seen in the heat stress time trials of EvaGreen dye, could be a result of volume differences in dye less than 0.25 L per reaction.

Field Conditions Trial

To evaluate the stability of EvaGreen exposed to field conditions, the dye was taken to Ali Al-Salem Air Force Base, Kuwait, for a 34-day deployment. In Kuwait, the dye was exposed to temperatures usually ranging from 19 Cto45 C, with a high temperature of 50 C noted. On return to the Air Force Academy, the dye was run alongside control samples stored at 4 C and compared to predeployment PCR results as well.

The mean Cq and end fluorescence values (with standard error) for 10 independent samples per group are shown in Figure 4. ANOVA by group revealed significant differences between groups, and comparison by Student's t-tests (Fig. 4) showed statistically significant differences in mean Cq values between the Day 0 group (20.67 0.21 Cq) and the dyes deployed to Kuwait (22.10 0.10 Cq) or stored at 4 C during that time. However, there were no significant differences between the mean Cq values of the deployed dye and the control dye stored at 4 C (21.98 0.13 Cq) during that time. Significant differences were found, however, between the mean fluorescence values of all three dye groups. Mean fluorescence was significantly lower in the deployed dye (9,934 511 FSU) and the 4C control (12,690 709 FSU) than it was during the Day 0 run (18,463 943 FSU).


Throughout the 176-day heat stress time trial, EvaGreen dye stored at 4C (control), 25C, 45C, and 65C did display statistically significant differences in mean Cq and end-point fluorescence. However, the differences in mean Cq were all less than one cycle and would thus be unlikely to have an effect on the overall outcome of a real-time PCR study. Although differences in mean end fluorescence as large as 2,113 FSU were found, levels were still well above 10,000 FSU and it is believed that such moderate decreases in fluorescence values would have little impact on the practical results of a real-time PCR run, especially as most investigators rely solely on Cq values. As evidenced by the volume trial, using as little as 0.25 L less dye than recommended per reaction created fluorescence differences larger than the statistically significant differences in means seen in the 176-day heat stress trial; this volume could be within the range of systematic and random error inherent to a pipette or retained in a pipette tip after blow out. Although the field condition trial did reveal significant differences in mean fluorescence, the lack of significant difference in Cq values between the dye that was deployed and the dye stored at 4 C demonstrates the stability of EvaGreen despite international travel and harsh field conditions.

In conclusion, our study demonstrates that liquid EvaGreen dye maintains effectiveness as a real-time PCR reagent when exposed to high temperatures for up to 6 months. The ability to use a single DNA-intercalating dye that does not require cold storage with multiple, customizable primer sets has the potential to greatly increase the applications of JBAIDS technology. Our results warrant further investigation by using liquid EvaGreen dye to adapt traditional lab-based real-time PCR assays for JBAIDS use and testing the assays in the field.


Funding and support was provided by the Air Force Medical Services Agency. Funds came from the Life Sciences Research Center yearly budget allotment for research.


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2d Lt Craig D. Nowadly, USAFR MC*; Cadet Jason W. David, USAF*; Melanie L. M. Grogger, MS*; Lt Col Erik R. Demkowicz, USAF (Ret.)*; Lt Col Daniel H. Atchley, USAF BSC (Ret.)[dagger]; Maj Donald V. Veverka, USAF (Ret.)*

*United States Air Force Academy, 2355 Faculty Drive, USAF Academy, CO 80840.

[dagger]Harding University College of Pharmacy, 915 East Market Street, Searcy, AR 72149.

The research described in this manuscript was presented orally by Cadet Jason David during the Colorado Springs Undergraduate Research Forum held at the University of Colorado Colorado Springs on April 13, 2013.

The views expressed in this article are those of the authors and do not reflect the official policy or position of the U.S. Air Force, the Department of Defense, or the U.S. Government.

doi: 10.7205/MILMED-D-13-00515

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Source: Military Medicine

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