OMNIgene®•DISCOVER stabilizes microbial DNA profiles in oral
fluid samples, enables more precise characterization of oral flora
A. Bouevitch, E. Doukhanine and C. Merino
DNA Genotek, Inc. Ottawa, Ontario, Canada

Abstract

Control

Complete linkage, correlation coefficient distancePowerful analytical methods like Microarrays and Next Generation Sequencing give researchers the ability to easily
detect and quantitate molecular targets in a highly multiplexed fashion. However, to take full advantage of such analyticalcapabilities, care must be taken to ensure that what is captured at the time of sample collection does not change by thetime samples are processed and analyzed.
Due to the complex nature of samples required for analysis of the oral and gut microbiome, bias can be easily introduced
through improper sample collection, stabilization and transport. In particular, the relative abundance of microbial species
or “microbial profile” can change rapidly in an non-stabilized sample. Likewise, integrity of nucleic acids can rapidly degrade
due to chemical and enzymatic activity.
DNA Genotek, in collaboration with the Forsyth Institute (developer of The Human Oral Microbe Identification Microarray(HOMIM)), designed this study to demonstrate the impact of standardized sample collection and stabilization. The HOMIM
array contains hybridization probes to the 16S rRNA gene sequence of around 300 of the most prevalent oral bacterial species
and has been shown to provide highly correlated results with 16S rRNA pyrosequencing at the phylum level, and is suitablefor high-throughput oral microbiome studies. Importantly, HOMIM is also lower cost and less labour intensive thansequencing (Ahn et al., 2011)1. In this study, untreated oral fluid samples were compared on the HOMIM array to oral fluidsamples collected and stabilized in OMNIgene•DISCOVER. The microbial DNA profiles of the untreated samples showedsignificant variation over the different temperatures and time points, whereas when stabilized with OMNIgene•DISCOVER
the microbial profiles remained constant regardless of storage time and temperature.

Similarity

75.31

Roomtemperature

83.54

37°C

91.77

TOMethods

T21

T5

T21

OM-505Complete linkage, correlation coefficient distance

Saliva from 10 healthy donors was collected into conical tubes. Equal volumes of saliva from each donor was transferred
into a 50 mL conical tube and mixed 10 times using a sterile pipette. 1 mL of the pooled saliva sample was transferred into(a) 9 Control tubes containing 1 mL of Saline and (b) 9 OMNIgene•DISCOVER OM-505 kits. For each set of samples 3 tubes
DNA was extracted immediately after collection, 3 tubes were incubated at room temperature and 3 tubes were incubatedat 37°C. DNA from these tubes was extracted after 5 or 21 days. Bacterial DNA from the Control tubes was extracted using
the protocol recommended by Dr. Bruce Paster, Forsyth Institute (HOMIM DNA Isolation Protocol). Bacterial DNA extraction
from OMNIgene•DISCOVER/saliva samples was done as follows: the whole sample was heated for 1 hour at 50°C followed
by heating for 15 minutes at 85°C; a 500 µL aliquot was transferred to a new 1.5 mL tube where it was disrupted using abead beater for 1 minute. The DNA was quantified using PicoGreen. Equivalent amounts of DNA from the control and theOMNIgene•DISCOVER/saliva samples were used for establishing the oral microbiome profiles on the HOMIM microarray.
The data from the HOMIM array was analyzed with Minitab 16 using a Cluster Analysis of Variables, based on complete
linkage and a final partition of 3 clusters.

Roomtemperature

Similarity

95.21

96.81

98.40

Room
temperature

100.00Control

T0

T537°C

Saliva sample

37°C

T5 T037°C

T21

T21 T21 T5
T5 T2137°C 37°C 37°C

T0

T5T5

T21 T2137°C 37°C

Figures 2a and 2b: Stabilization reduces variability in sample after collection.
HOMIM
array

OMNIgene•
DISCOVER

Extraction

T5T= 0Roomtemperature

Roomtemperature

37°C37°C

T= 5 days

T= 21 days

Detection

Conclusion
The benefits of optimized sample collection and stabilization with OMNIgene•DISCOVER:• The ability to collect and stabilize ‘microbial profiles’ over time, even at elevated temperatures• Reduces bias. Efficient recovery/detection of nucleic acids from both gram positive and gram negative bacteria• Applicable to research on microbiome, dentistry, and infectious diseases• Cost effective, reliable self-collection by untrained users – increase participation and ensure study protocol compliance• Standardized sample collection ensures samples collected at different times/locations within a study can bemeaningfully compared.

14 daysFigure 1: Experimental workflow.
OM-505Control

T= 5 days

Results
After extraction, the integrity of the DNA was assessed byagarose gel. DNA extracted from all samples showed a single,
high molecular band (data not shown). After quantification using
PicoGreen® (Invitrogen), similar amounts of DNA were used
for the establishment of microbial DNA profiles using HOMIM
microarray. Cluster Analysis was used to analyze the data.
The most relevant findings are:
• The similarity between the triplicates of the non-stabilized
control samples processed immediately after collection
was 89.09%. This represented the inherent variability of theHOMIM microarray. Triplicate control samples for all conditions
presented a comparable or higher similarity levels than thecontrol at time 0.
• The statistical analysis (Cluster Analysis of Variables) determinedthat samples incubated at room temperature or at 37°C aregrouped in the same cluster, irrespective of the incubation
time (similarity = 83.9% and 87.6% respectively). The similarity
between the time 0 Control and these clusters were 79.4%(room temperature) and 75.3% (37°C) (Figure 2a). These results
suggested that without proper stabilization the microbial DNA
profile is significantly impacted by time and environmental
conditions, most likely by differential bacterial growth andsurvival.
• In contrast to the non-stabilized control samples, clustering
among samples collected and stabilized in OMNIgene•DISCOVER
OM‑505 is not based on storage time or temperature. Similarity
among all samples was 95% (Figure 2b). Of note, one cluster
contained a sample processed immediately after collection anda sample processed after 21 days at 37°C. This clearly
demonstrates that collection and stabilization inOMNIgene•DISCOVER negates the effects of time andtemperature on microbial profiles and DNA integrity.

Probe

T= 0

RT

T= 21 days
37°C

RT

T= 5 days
37°C

T= 0

RT

T= 21 days
37°C

RT

37°C

Buffer
Negative Control
Positive Control_16S_Universal_E29
Capnocytophaga granulosa and sp clone BB167_ot325_326_AA89
Capnocytophaga sputigena_ot775_W46
Prevotella histicola_ot298_AD71
Prevotella melaninogenica and sp clone BE073_ot298_469_T81
Prevotella nigrescens_ot693_W40
Prevotella oralis_ot705_X76
Prevotella pallens and sp clone DR022_ot310_714_R67
Prevotella sp clone DO039_ot308_Q94
Prevotella sp clone DO045_ot309_Q97
Prevotella Cluster IV_ot658_693_714_782_AA44
Tannerella forsythensis_ot613_X56
Haemophilus sp clone BJ095_ot036_AA97
Campylobacter gracilis_ot623_X34
Campylobacter showae_ot763_X35
Neisseria gonorrhoeae and polysaccharea_ot621_737_O76
Dialister pneumosintes_ot736_X78
Megasphaera micronuciformis_ot122_AA57
Megasphaera micronuciformis_ot122_X28Solobacterium moorei_ot678_AC01
Solobacterium moorei_ot678_AC02
Parvimonas micra_ot111_L97
Parvimonas micra_ot111_V05
Lachnospiraceae[G-3] sp clone DO008_ot096_AA70
Lachnospiraceae[G-4] sp clone DO016_ot097_R64
Eubacterium[11][G-6] minutum_ot673_AC65
Eubacterium[14][G-1] saburreum and Lachnospiraceae[G-1] sp
Streptococcus anginosus and intermedius_ot543_644_Q62
Streptococcus constellatus and intermedius_ot576_644_AB77
Streptococcus constellatus and intermedius_ot576_644_F48
Streptococcus mitis bv2 and sp clone FP064_ot069_398_Q64
Leptotrichia buccalis and goodfellowii and Sneathia sanguinegens
Rothia dentocariosa and mucilaginosa_ot587_681_E52
Rothia mucilaginosa_ot681_AB62
Rothia mucilaginosa_ot681_AB63
Veillonella EF509966 Crohn's_not_oral_AD63-C

Figure 3: Stabilization of microbial DNA profiles vs. control samples at room temperature (RT) and 37°C over 0, 5, and 21 days.

“By using HOMIM (Human Oral Microbe Identification Microarray), we demonstrated that saliva samples stored in OMNIgene
were indeed stabilized for at least 3 weeks, even at 37 degrees. This is outstanding! Consequently, I think OMNIgene would bevery useful for remote collections alleviating the need for a -80 freezer or, for that matter, any refrigeration to preserve samples.
This is an incredible benefit for any investigator who is interested in preserving precious clinical samples, especially for use
with molecular analyses of genomic material.”
Bruce J. Paster, Ph.D., Director, Human Microbe Identification Microarray Core
Head, Department of Molecular Genetics, The Forsyth Institutehttp://mim.forsyth.org

Reference
1 Ahn JY, Yang LY, Paster BJ, Ganly I, Morris L, Pei ZH, Hayes RB. Oral microbiome profiles:
16S rRNA pyrosequencing and microarray assay comparison. PloS One2011;6(7):e22788

Superior samples
Proven performance

European Patent No. 1 956 969; Patent pending
Canadian Design Nos. 127470; 132896; 132897
U.S. D631,554 S and D640,795 S
Community Design Nos. 001095186-0001; -0002; -0003
OMNIgene®•DISCOVER is for research use only, not for use in diagnostic procedures.
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MK-00090 Issue 1/2012-06 © 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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