Evaluation of Swabs, Transport Media, and Specimen Transport
Conditions for Optimal Detection of Viruses by PCR
Julian Druce, Katherine Garcia, Thomas Tran, Georgina Papadakis, and Chris Birch
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs col-
lected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection
of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for
7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large num-
bers of cases.
D uring the 2009 influenza A H1N1 pandemic, the number of
samples referred for influenza virus testing to our laboratory
increased substantially (3). Virological swabs and transport me-
dium became in short supply, and alternatives were often used,
although systematic data regarding their suitability were lacking.
Molecular detection methods do not require replication com-
petent virus, but preservation of nucleic acid is essential. Previous
studies have examined the swab types (1, 6, 9, 10) and transport
medium (7) optimal for identification of viruses, but often they do
not assess variables such as transit times and holding temperatures
between sample collection and testing. While many environmen-
tal studies have investigated the effects of temperature on virus
survivability (2, 4, 5, 8), less is known about the impact of temper-
ature on nucleic acid detection by PCR.
This study examined the stability of four viruses with different
physicochemical properties in several swab and transport me-
dium combinations. The viruses, each of which were isolated in
our laboratory, were influenza A/New Caledonia/20/99 (H1N1)
and an enterovirus (echovirus type 30), representing enveloped
and nonenveloped RNA viruses, respectively; and herpes simplex
virus type 2 (HSV-2) and adenovirus type 7, representing envel-
oped and nonenveloped DNA viruses, respectively. We examined
the impact of swab type, viral transport medium, and the duration
and temperature of transport.
Swabs were purchased from Copan (Brescia, Italy). The types
of swabs were as follows: plain (rayon-tipped) swabs without me-
dium (catalogue no. 8154CIS), virus transport medium swabs in a
medium-soaked sponge (VTM) (catalogue no. 147CV), flocked
swabs with universal transport medium (UTM) (catalogue 359C),
flocked nylon fiber swabs with liquid Amies medium (E swabs)
(catalogue 480CE), and swabs with Amies gel containing charcoal
(catalogue E114).
Virus stocks were prepared in minimum essential medium
(MEM) (Sigma, Manheim, Germany) containing 2% fetal bovine
serum (FBS) (Gibco, Auckland, New Zealand) (MEM2%) to give
cycle threshold (CT) values of between 22 and 25 (approximately
10,000 to 1,000 nucleic acid copies per ml, respectively). Swabs
were immersed in the virus for 2 s, and those with associated
transport media were immediately placed in that medium. Amies
gel swabs were resheathed in the gel. Plain swabs were either
resheathed in their original housings without added medium
(dry) or placed in a tube containing 2 ml sterile saline. Duplicates
of each swab type exposed to the individual viruses were immedi-
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ately stored at ⫺70°C as time zero controls. The remaining swabs
were divided into 3 groups and held at 4°C, ambient temperatures
(20 to 22°C), or 37°C. Duplicate swab/medium combinations
from each treatment condition were then collected and stored at
⫺70°C every 24 h for 7 days. All swabs were incubated for a further
7 days at ⫺70°C and then thawed at 4°C overnight. The medium
on VTM, UTM, E, and saline swabs was used directly for nucleic
acid extraction (the volume in VTM swabs was increased by 2 ml
with MEM2%). Plain swabs without transport medium (dry) and
the Amies gel swabs were placed in 2 ml MEM2% immediately
prior to extraction.
Total nucleic acid in a 200-␮l volume of sample was extracted
using Qiagen DX extraction kits (Qiagen Sciences, Germantown,
MD) and a QIAxtractor robot (Qiagen, Hilden, Germany). The
elution volume was 60 ␮l. To obtain cDNA, 10 ␮l extract was
linearized at 65°C for 10 min, quenched on ice, added to 12 ␮l
reverse transcription (RT) master mix containing random hex-
amers (Roche Diagnostics, Mannheim, Germany), deoxynucleo-
side triphosphates (dNTPs) (Amersham Biosciences, Bucking-
hamshire, United Kingdom), and avian myeloblastosis virus
(AMV)-RT enzyme and buffers (Promega, Madison WI), and
then incubated for 30 min at 42°C and 10 min at 100°C.
PCR used 4 ␮l DNA and 16 ␮l ABI Fast master mix (Applied
Biosystems, Foster City, CA) containing primers at 0.9 ␮M and
probes at 0.2 ␮M (sequences available on request). Cycling con-
ditions were 95°C for 2 min then 45 cycles of 95°C for 2 s and 60°C
for 30 s using an ABI 7500 fast real-time system (Applied Biosys-
tems, CA).
CT values obtained for influenza virus under the conditions
evaluated are shown (Fig. 1). Use of UTM, VTM, and E was con-
sistently associated with an amplifiable product. Plain swabs in
saline also yielded an amplifiable product at each time point and
temperature, with CT levels similar to those of the commercial
products. Using linear regression, there was a statistically signifi-
cant difference in CT over time between saline swabs held at either
Received 30 November 2011 Accepted 16 December 2011
Published ahead of print 28 December 2011
Address correspondence to Julian Druce, julian.druce@mh.org.au.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.06551-11
0095-1137/12/$12.00 Journal of Clinical Microbiology p. 1064 –1065
 Optimal Swabs and Transport Media for Viral PCR

from Amies gels were consistently higher for these 3 viruses. This
was not unexpected, since this product has been developed for
preservation of anaerobic bacteria, but is sometimes used to col-
lect and transport specimens for viral studies.
This study differed from several previous investigations that
compared swab types and specimen collection methods within a
clinical setting (1, 6). Our approach was laboratory based, and no
patients were involved. Although CT values provide some quanti-
tative information, issues such as the relationship between viral
load and disease severity could therefore not be assessed. A study
similar to ours has shown that respiratory syncytial virus (RSV)
could be detected using PCR on dry cotton swabs after 15 days at
room temperature (9). In the same study, dry swabs used for
clinical sampling showed utility for several respiratory viruses,
although no direct comparison to other transport media was

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made (9).
We did not evaluate the impact of swabs, transport media, or
transport conditions on virus isolation, although it is unlikely that
isolation would match the sensitivity of PCR analysis under the con-
ditions chosen. However, reference laboratories need to be mindful
that during outbreaks representative virus isolates are sometimes
needed for purposes such as strain characterization, supply of candi-
date vaccine strains, and drug resistance phenotyping.
The utility of swab/medium combinations for the molecular
detection of influenza virus is of considerable importance. This
virus is historically associated with large epidemics and is more
likely than other viruses to cause depletion of commercially avail-
able stocks of swabs and other consumables during times of high
testing demand. Our observation that collection and transport of
influenza virus on dry swabs in saline is appropriate for PCR de-
tection is important for future pandemic planning.

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FIG 1 Detection of influenza virus RNA by real-time PCR analysis from swabs
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