Stability, recovery efficiency and detection limit of viral DNA
in OMNIgene®•ORAL (OM-505)
E. Doukhanine and C. Merino

DNA Genotek, Ottawa, Ontario, Canada

OMNIgene®•ORAL (OM-505) is designed to collectand stabilize microbial DNA and RNA from oralfluids. In this document, we demonstrate thatOMNIgene•ORAL stabilizes viral DNA for up to21 days at room temperature. We also show that viral
DNA collected and stabilized in OMNIgene•ORAL
does not adversely affect detection of viral DNA
by downstream applications such as PCR.

IntroductionAccurate detection of viruses and bacteria bymolecular techniques greatly depends on the quality
and quantity of the sample after collection. Poorly
preserved samples may provide misleading results
including false negatives or false positives. Such bias
may result in misdiagnosis, improper selection and/or dosage of therapies (e.g., antibiotics or antivirals),
and may skew the results of epidemiology studies.
Typical sources of error in samples that are notproperly stabilized and preserved are:••••Selective overgrowth of a subset of microorganismsFormation of spores
Chemical and enzymatic degradationof nucleic acids
Impaired ability to detect targets oflower abundance

Current strategies to mitigate these stability
challenges include storing and transporting samples
at low temperatures, accelerating turn-around times
during processing and drying the samples over solid
matrix (i.e., paper). These strategies are expensive,
impractical and in some cases inefficient.
Preserved samples must yield reliable results from avariety of downstream applications, especially whenthe microbe of interest is present in low quantities.
Therefore, preservation methods must not introduce
inhibition.

OMNIgene•ORAL is an all-in-one system for thecollection and stabilization of microbial DNA andRNA from oral fluids. The kit stabilizes nucleic acids
at ambient temperature, enabling cost-effective
transportation over prolonged time and distance.
This method allows for the painless and non-invasive
collection of high quality samples that facilitate nucleic
acid detection from DNA and RNA viruses andbacteria, without inhibition, even in low copy numbers,
across a variety of molecular methods (e.g., RT-qPCR
or the Abbott m2000rt RealTime System).
This technical bulletin describes the stability andrecovery efficiency of viral DNA spiked into oral fluid
samples collected with the OMNIgene•ORAL kit.
Inactivated Epstein Barr-Virus (EBV) was used as atarget for detection using the Corbett Life Sciences
Rotor-Gene™ instrument. In-house PCR protocol wasused to analyze EBV DNA from spiked oral fluid
samples collected in OMNIgene•ORAL (OM-505).

Materials and methods
Inactivated EBV virus (NATtrol EBV, Zeptometrix)
was used in all experiments. NATtrol EBV is preparedfrom highly purified virus, isolated from cell culture,which is chemically and enzymatically treated to alter
its surface proteins rendering it unable to infect ahost cell. While modified on its surface, the virus
still retains an intact and complete viral genome,
making it an ideal control1.
Purified viral DNA from NATtrol EBV was used
to optimize and validate the conditions for aquantitative PCR (qPCR). Serial dilutions of
EBV DNA were tested in triplicate with custom
designed primers for the LMP-2A EBV gene
(Forward: 5’-CATGTTCTGGGTCAAAAGG andReverse: 5’-TGCTGATTCTGCTAACACTG).
PCR reaction: in a 25 μL reaction volume; 0.2 U TaqPolymerase, 200nM primers, 1.5 mM MgCl2,

For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.
PD-WP-00028 Issue 2/2015-01© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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1 μM Syto®9 dye (Invitrogen), PCR Buffer without
MgCl2 (Roche), 0.1 mg/mL BSA. PCR conditions
were used: initial denaturation for 5 minutes at 95°C
(1 cycle); denaturation for 30 seconds at 95°C,annealing 40 seconds at 58°C and extension for
30 seconds at 72°C (40 cycles), final extension for
7 minutes at 72°C. All qPCR runs were performedon the Corbett Life Sciences Rotor-Gene™ and thedata was analyzed using the default quantitation
analysis with the built-in software, Rotor-Gene™ 6000
ver. 1.7. The qPCR assay resulted in a 4 log linear
dynamic range (80 - 20,000 copies cp of EBV virus
per reaction), a reaction efficiency of 1.01 ±0.07,
R2 of 0.98, no detectable amplification in theno-template control and a single amplified productwith the expected theoretical melting temperature.

Results“Sample” in this document refers to oral fluids
collected in OMNIgene•ORAL per kit instructions.
To determine the limit of detection (LOD) of theassay, samples were spiked with known amounts of
EBV NATtrol: very low copy: 320 cp/mL; low copy:
1,600 cp/mL; medium copy: 3,200 cp/mL; highcopy: 16,000 cp/mL. To determine the ability ofOMNIgene•ORAL to stabilize viral nucleic acids,samples were spiked with 3,200 cp/mL of EBVNATtrol. Samples were then incubated at room
temperature (22°C to 24°C) for 0, 7 and 21 days
prior to purification. In all cases, viral DNA wasextracted from a sample aliquot of 250 µL using
the Qiagen QIAamp Virus MinElute Spin Kit,
then immediately frozen until ready to use.
EBV DNA was detected using the EBV qPCR
protocol described above.

2The ΔCq method2 was used to evaluate the extraction
efficiency and possible changes over time in EBV DNAcontent. The reference Cq value for the experimental
control was obtained using an equivalent amountof purified EBV DNA (standard curve). Due toinherent variability in qPCR amplification, a ΔCq
threshold of ±1 cycle was used, whereby any samples
demonstrating more than 1 cycle difference between
sample and standard was considered significant.

After OMNIgene•ORAL/oral fluid samples werespiked with either very low, low, medium or highquantities of EBV NATtrol, they were extracted andquantified as described above. All data were analyzed
using the ΔCq method (Figure 1). In all cases, lessthan 1 cycle difference was observed between thecontrol and EBV spiked samples. Greater variability
among the very low EBV spiked sample group wasobserved as compared to other groups; however thedetection was still within the established threshold.
These results indicate that viral DNA from oral fluids
collected with OMNIgene•ORAL can be reliably
amplified, even in samples with a very low viral load.

EBV DNA detection∆Cq10-1-2

4000cp

800cp

400cp

80cp

EBV spiked in sample(Estimated EBV copies/reaction)
Figure 1: qPCR detection of EBV DNA in spiked OMNIgene•ORAL/oralfluid samples. ΔCq = Cqsample - Cqcontrol. ΔCq values are presented
as a mean of 3 experimental replicates with standard error of mean.

Spiked samples were stored at room temperatureand viral DNA was extracted at T= 0, T= 7 days or
T= 21 days. No differences in the recovered amountof EBV viral DNA were observed between thesamples processed 0, 7 or 21 days after collection(Figure 2). These results indicate that viral DNA inoral fluid samples collected with OMNIgene•ORAL
is stable for up to 3 weeks at room temperature.

EBV DNA stability inOMNIgene•ORAL/saliva sample

For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.

∆Cq

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EBV DNA stability inOMNIgene•ORAL/saliva sample∆Cq

1Conclusions
1. OMNIgene•ORAL (OM-505) enables successful
PCR amplification of even very low viral loads ofa human DNA virus.
2. OMNIgene•ORAL (OM-505) stabilizes viral
DNA from human viruses in oral fluid whenstored for up to 21 days at room temperature.

0-1
Day 0Day 7Day 21Figure 2: Room temperature stability of EBV DNA in OMNIgene•ORAL/oral fluid samples. ΔCq values are presented as a mean of 3 experimental
replicates with standard error of mean; 800 cp/reaction.

References:1

http://www.zeptometrix.com/nattrol.htm

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®OMNIgene is a registered trademark of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.
PD-WP-00028 Issue 2/2015-01© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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