![PrimeStore MTM inactivates nucleases and preserves (a) influenza A viral RNA and (b) single-stranded internal positive control (IPC) RNA. Real-time RT–PCR analysis was performed using : 10 3 TCID 50 /ml whole influenza A(H3N2) virus preserved in PrimeStore MTM and other media* (a), and IPC RNA (b) after incubation in RNA/DNA nucleases (Ambion RNase A, T1, and Turbo DNase) at 37 xC for 48 h. Nucleic acid extraction at 1, 24 and 48 h was performed and PCR amplified using PrimeMix Universal Influenza A and PrimeMix IPC assays (Longhorn Vaccines & Diagnostics). Mean and standard error bars for duplicate extractions are shown. A C T reading of 40 (C T =40) is no amplification. [* Viral transport media (VTM) is BD Universal Viral Transport Medium for Viruses, Chlamydiae, Mycoplasmas and Ureaplasmas (BD, USA) ; AVL Lysis Buffer is Buffer AVL viral lysis buffer (Qiagen, cat. no. 1014777 ; USA). ZR Viral RNA Buffer is from Zymo Research (cat. no. R1034-1-50).]](https://www.researchgate.net/profile/Rick-Schuman/publication/49727970/figure/fig1/AS:342073304731649@1458568039673/PrimeStore-MTM-inactivates-nucleases-and-preserves-a-influenza-A-viral-RNA-and-b_Q320.jpg)
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A clinical specimen collection and transport medium for
molecular diagnostic and genomic applications
L. T. D AUM
1
*, S. A. W O RTH Y
1
,K.C.YIM
2
, M. NOGUERAS
3
,R.F.SCHUMAN
4
,
Y. W. C HOI
5
AND G. W. F ISC H ER
1
1
Longhorn Vaccines & Diagnostics, San Antonio, TX, USA
2
Virion Systems Inc., Bethesda, MD, USA
3
BioReliance Corp., Rockville, MD, USA
4
Antibody and Immunoassay Consultants, LLC, Rockville, MD, USA
5
Battelle Biomedical Research Center, Columbus, OH, USA
(Accepted 30 September 2010)
SUMMARY
Pathogen detection and genetic characterization has dramatically changed in recent years.
Clinical laboratories are transitioning from traditional culture and primer-specific sequencing
to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic
characterization, respectively. Specimen collection is the first step in any downstream molecular
diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of
nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be
amplified using real-time RT–PCR for tracking the integrity of a specimen from the point of
collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including
highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released
RNA at ambient temperature for up to 30 days for downstream real-time and traditional
RT–PCR detection and genetic characterization. PrimeStore MTM is also compatible with a
variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has
added utility for field collection in remote areas, triage centres, border crossings and during
pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens
are a concern.
Key words: Influenza detection, Longhorn Vaccines and Diagnostics, molecular diagnostics,
molecular transport medium, PrimeStore MTM, real-time RT–PCR, specimen collection and
storage, viral transport media.
INTRODUCTION
Emerging infectious respiratory diseases constitute
a continuing public health threat worldwide. Rapid
movement of people and goods around the world
increases the opportunity for local outbreaks to quickly
become worldwide pandemics. The 2009 H1N1 swine
influenza pandemic [1, 2] and continuing circulation
and evolution of H5N1 avian influenza isolated from
humans [3, 4] underscores a need for safe specimen
collection kits that : (1) are compatible with molecular
diagnostic and genomic analysis, (2) can effectively
inactivate potentially harmful microbes, (3) stabilize
and preserve nucleic acids including RNA, and (4) can
* Author for correspondence : L. T. Daum, Ph.D., Chief Scientific
Officer, Longhorn Vaccines & Diagnostics, 1747 Citadel Plaza,
Suite 206, San Antonio, Texas 78209, USA.
(Email : Longhorn-VandD@sbcglobal.net)
Epidemiol. Infect., Page 1 of 10. fCambridge University Press 2010
doi:10.1017/S0950268810002384
be easily used in the field and transported at ambient
temperatures while preserving microbial RNA.
Until recently, the majority of clinical diagnostic
laboratories employed traditional culture for patho-
gen identification that typically requires 3–7 days for
most viruses [5] and longer for some bacterial strains
[6]. Traditional culture requires specimen collection of
viable microbes, frozen transport, propagation and
handling of potentially infectious and often unknown
biological microbes. Furthermore, many infectious
agents, e.g. highly pathogenic avian influenza, SARS,
etc., are BSL-3 level pathogens that require specia-
lized facilities and precautions for analysis. There are
challenges in obtaining, shipping and maintaining
high-quality, viable biological specimens for culture.
Specimens must be shipped using a cold chain, most
often dry ice. Transporting potentially infectious
samples from remote sites or across international
borders using commercial transit can be costly and
tedious, particularly when specimens must be received
frozen.
The field of clinical molecular diagnostics changed
drastically with the advent of polymerase chain re-
action (PCR) [7], and subsequently, real-time PCR [8].
Real-time reverse transcription–PCR (rRT–PCR) can
deliver superior sensitivity and specificity results in
hours [9]. Thus, the majority of current diagnostic
laboratories have transitioned from traditional culture
to nucleic acid testing (NAT) such as real-time PCR.
Collection is the first step in diagnostic platforms or
molecular protocols requiring the detection of poten-
tially minute amounts of nucleic acids from microbes.
Regardless of the nucleic acid test used or the
RNA/DNA extraction protocol, specimen collection,
specifically the inactivation of potentially infectious
agents and the preservation and stability of pathogen
RNA/DNA remains a critical gap in clinical diag-
nostics, especially for use around the world.
This work describes the use of PrimeStore Mol-
ecular Transport Medium (MTM ; Longhorn Vaccines
& Diagnostics, USA), a clinical or environmental
sample collection system specifically formulated for
downstream molecular diagnostic testing. PrimeStore
MTM is an optimized blend of proprietary reagents
for RNA preservation that also includes the first non-
specific exogenous internal positive control (IPC) for
tracking the integrity of a specimen from the point
of collection to detection. PrimeStore MTM is shown
to efficiently: (1) lyse/inactivate potentially infectious
biological pathogens reducing infection risk so that
samples and can be handled, shipped, or transported
with minimal fear of pathogen release or contami-
nation, (2) stabilize and protect lysed ‘ naked’ RNA
polymers from hydrolysis, oxidative damage or
nuclease degradation, (3) preserve RNA for prolonged
periods at ambient temperature until they can be
processed using NAT, and (4) be compatible with
commercially available bench-top extraction kits.
MATERIALS AND METHODS
Microbe killing
Membrane filtration technique for bacterial and
fungal recovery
The membrane filtration method for bacterial and
fungal recovery was used to assess the killing ability
of PrimeStore MTM. Escherichia coli,Pseudomonas
aeruginosa,Staphylococcus aureus [non-methicillin-
resistant Staphylococcus aureus (MRSA)], Candida
albicans,Bacillus subtilis,andAspergillus brasiliensis
were used to determine whether PrimeStore MTM
could effectively kill and inactivate a panel of bacteria
and mould (yeast and filamentous fungi). Positive
controls incubated in a water matrix were performed
on day 0 only. A population of 1r10
6
c.f.u. for each
bacterial strain was inoculated into 0.5 ml PrimeStore
for each time-point and subsequently incubated
at 20–25 xC. The containers were enumerated and
evaluated at days 0, 1, 7, 14 and 28. The inoculum was
aseptically passed through a sterile filtration device
and subsequently rinsed three times with 100 ml
sterile neutralizing fluid D [1 g peptic digest of animal
tissue (peptone) and 1 ml polysorbate 80 dissolved
in 1.0 l of sterile water (final pH 7.1¡0.2)]. Where
necessary, dilutions of the inoculated test article were
performed to deliver a target count of 25–250 c.f.u.
per filter. For each time-point, inoculated negative
controls were processed in a similar fashion. Filters
inoculated with samples containing bacteria were
plated onto tryptic soy agar (TSAP) with lecithin and
polysorbate 80 and incubated at 30–35 xC for 72 h.
Filters inoculated with samples containing yeast or
mould were plated onto Sabouraud dextrose agar
(SAB) and incubated at 20–25 xC for no less than 72 h
but no more than 5 days. Colonies were counted to
calculate log
10
recoveries and percent (%) kill for each
organism used during microbial challenge.
Viral killing
Equal volumes of A/Wuhan/359/95 (10
8
TCID
50
/ml)
or adenovirus (type 5) (10
8
TCID
50
/ml) and
2 L. T. Daum and others
PrimeStore MTM were incubated for 10-, 30-, and
60-s intervals at ambient temperature. Influenza virus
only and PrimeStore MTM (minus virus) were also
tested as controls. After incubation, each solution was
subjected to a tenfold serial dilution, and inoculated
onto a 96-well plate confluent with MDCK cells
(influenza) or A549 (adenovirus). Prior to influenza
virus or adenovirus inoculation, 96-well plates
were washed 2rwith EMEM or EMEM containing
1mg/ml trypsin, respectively. All samples were pro-
cessed in quadruplicate. Plates were incubated at
37 xC with 5% CO
2
for 4 days, stained with Crystal
Violet reagent (0.06%) in 1% glutaraldehyde for 1 h
and rinsed with sterile water. Cells not stained ex-
hibited cytopathic effect (CPE) of virus. The titre of
the virus was recorded as the TCID
50
, i.e. the inverse
of the dilution that resulted in the CPE in 50 % of
the wells. Viral titres were calculated as geometric
means+standard error.
For testing against influenza A/Vietnam/1203/
04(H5N1) virus (10
7
TCID
50
/ml), a collection swab
was loaded with 0.1 ml H5N1 virus and placed in
1.5 ml PrimeStore MTM. Positive controls consisted
of virus-loaded swabs in the absence of PrimeStore
MTM, and swabs in PrimeStore MTM without
influenza virus were used as negative controls.
After 60-min incubation at ambient temperature, the
samples were vortexed and inoculated into 10-ml cell
culture media. Samples were serially diluted (tenfold)
and inoculated onto 96-well plates confluent with
MDCK cells. Plates were incubated at 37 xCwith5%
CO
2
for 4 days and assessed for viral CPE.
MRSA killing
A stock plate containing about 10
8
c.f.u. MRSA
(ATCC 33592) was transferred to TSB, vortexed
briefly and incubated at ambient temperature for
10 min. A total of 0.1 ml bacterial suspension was
transferred to 0.9 ml PrimeStore and vortexed for
60 s. A total of 0.1 ml suspension was transferred to
0.3 ml TSB (1 :4 dilution) and 100 ml was transferred
to blood agar plates (5 % sheep RBCs in TSA) after 0,
5 and 15 min. Positive controls included equivalent
volumes of MRSA and TSB. Plates were allowed to
dry, incubated overnight at 37 xC and analysed for
c.f.u./ml.
Nuclease digestion experiment
Stock influenza A(H3N2) virus (10
3
TCID
50
/ml)
spiked into human nasal washing was preserved in
PrimeStore MTM or other indicated media (0.3ml
solution to 0.1 ml virus) and incubated at 37 xCwith
RNase A (25 U), RNase T1 (125 U) and Turbo
DNase (2 U) for 1, 24 and 48 h (Life Technologies
Inc., USA). Reactions were inactivated at 75 xC for
2 min prior to RNA extraction.
Test samples and RNA extraction
For the RNA stabilization and preservation exper-
iments described in Figures 2 and 3, a human throat
swab containing a respiratory oral/pharyngeal matrix
was placed into collection tubes containing 1.5ml
PrimeStore MTM and viral transport medium (VTM ;
BD Universal Viral Transport Medium ; USA). An
influenza culture containing 10
2
TCID
50
/ml (Fig. 2)
or 10
4
TCID
50
/ml (Fig. 3) of A/California/04/2009
influenza A(H1N1) was spiked into each PrimeStore
MTM or VTM tube and incubated at the indicated
temperature (4, 25 or 37 xC).
RNA was extracted using the Ambion RNaqueous-
Micro kit (Life Technologies) according to the manu-
facturer’s protocol. For PrimeStore compatibility
experiment with commercial extraction kits (Fig. 4),
the Ambion RNAqeuous-Micro (Ambion cat. no.
1931), QIAamp Viral RNA Mini kit (cat. no. 52904),
Invitrogen Charge Switch Total RNA Cell kit
(cat. no. CS14010), and Ambion MagMAX Viral RNA
Isolation kit (cat no. 1928) were utilized according to
the manufacturer’s recommendations with 100 mlof
10
3
or 10
1
TCID
50
/ml A/California/04/2009 influenza
A(H1N1) stock culture. All time-point extractions
were frozen at x25 xC until analysis.
Real-time and traditional RT–PCR
For rRT–PCR, a 1rmaster-mix (PrimeMix) con-
taining real-time primers and probes specific for the
universal detection of influenza A or an IPC RNA
present within PrimeStore was utilized for detection
according to the manufacturer’s recommendations
(Longhorn Vaccines & Diagnostics, USA). Amplifi-
cation was performed using an ABI 7500 instrument
(Life Technologies). RT–PCR thermocycling con-
sisted of an RT incubation step at 50 xC for 20 min
followed by hot-start activation at 95 xC for 5 min
and 40 amplification cycles at 95 xC for 15 s and 60 xC
for 32 s.
Traditional RT–PCR was performed using the
SuperScript III High Fidelity One-Step kit (cat.
no. 12574-035, Invitrogen, USA) according to the
manufacturer’s recommendations. Two primer pairs
A molecular transport medium for nucleic acid testing 3
(forward 1204: 5k-TGTAAAACGACGGCCAGTAA-
GATGAAYACRCARTTCACAG-3kand reverse 1778:
5k-CAGGAAACAGCTATGACCGTGTCAGTAGA-
AACAAGGGTGTTT-3kand forward 379: 5k-TGT-
AAAACGACGGCCAGTACRTGTTACCCAGGR-
GATTTC-3kand reverse 1204 : 5k-CAGGAAACA-
GCTATGACCTCTTTACCYACTRCTGTGAA-3k)
described by the World Health Organization [10]
for amplification and sequencing influenza A(H1N1)
2009 field strains were used to generate a fragments
with base-pair (bp) lengths of exactly 574 and 825 bp,
respectively, from the H1N1 influenza haemag-
glutinin gene. Amplification was performed using
an Applied Biosystems 2720 instrument (Life Tech-
nologies). RT–PCR thermocycling consisted of an
RT incubation step at 50 xC for 30 min followed
by hot-start activation at 95 xC for 2 min and 40
amplification cycles at 95 xC for 15 s, 52 xC for 15 s,
and 68 xC for 1 min. A final incubation of 68 xC
for 5 min for extension was performed. Influenza
haemagglutinin gene fragments were visualized by
UV illumination using 1.2% pre-made agarose gels
stained with ethidium bromide (Invitrogen).
RESULTS
PrimeStore MTM microbial and viral inactivation
Microbial inactivation
PrimeStore MTM was shown to rapidly inactivate
microbes including fungi, Gram-positive/-negative
bacteria and viruses. Certified USP 32-NF 27<51>
Antimicrobial Effectiveness Testing was performed
using the membrane filtration technique for the
quantitation of bacteria and fungi. At the first test
period (24 h), 100 % of bacteria and fungi were killed
compared to the positive controls (Table 1 a). For
these microbes, PrimeStore MTM met the inacti-
vation criteria as described in USP Category 1 pro-
ducts (injections, emulsions, optic products, sterile
nasal products, and ophthalmic products made with
aqueous bases or vehicles). Additionally Bacillus sub-
tilis spores were challenged using the method descri-
bed in USP 51 to further evaluate PrimeStore MTM
inactivation of microbial populations. B. subtilis
spores were reduced by 99% within 24 h of exposure
(Table 1a). In a time-kill study of MRSA inoculated
into PrimeStore MTM, viable bacteria were not de-
tected (100% killing) at the earliest study time (5 min
post-inoculation) or at any of the later evaluation
times.
Viral inactivation
No viable virus influenza A(H3N2) or adenovirus
type 5 was detected within seconds after being placed
into PrimeStore MTM using viral loads as high as
10
7
–10
8
TCID
50
. The recovery of highly pathogenic
influenza A(A/Vietnam/1203/04) H5N1 virus at 10
8
TCID
50
/ml after incubation in PrimeStore MTM
was equivalent to negative controls, indicating com-
plete viral inactivation of H5N1 viruses (Table 1 b).
Because PrimeStore MTM is designed to destroy
membranes of cells and microbes, both MDCK and
A549 cell lines were destroyed until the test samples
were diluted 1 :1000, LOD 10
3
. Therefore, >4 log re-
ductions (>99.99% killing) were documented in all
spiked samples placed in PrimeStore MTM (Table 1b).
Table 1. PrimeStore Molecular Transport Medium
inactivates : (a)bacteria, fungi and (b)viruses
(a) Bacteria and fungi
Organism*
Positive
control
(c.f.u./ml)
PrimeStore+
organism
(% killed)
E. coli 6.4r10
7
100
P. aeruginosa 3.6r10
7
100
S. aureus 6.0r10
7
100
C. albicans 5.7r10
7
100
A. brasiliensis 1.9r10
6
100
B. subtilis (spores) 3.7r10
6
99.9
MRSA 4.7r10
8
100
(b) Viruses
Organism#
Positive
control
(TCID
50
/ml)
PrimeStore+
organism
(% killed)$
Influenza A(H3N2) 7.5r10
8
>99.99
Adenovirus type 5 7.7r10
8
>99.99
Influenza A(H5N1) 1.0r10
7
>99.99
* All bacteria with the exception of MRSA were tested
using the Membrane Filtration Technique. MRSA killing
was performed using standard c.f.u. plate enumeration
method.
#Influenza A(H3N2) inactivation testing performed by
Virion Systems Inc. Highly pathogenic influenza A(H5N1)
inactivation testing performed at Battelle Biomedical
Research Center.
$Initial 4-log dilutions of PrimeStore MTM+viruses were
required prior to inoculation into tissue culture lines due to
PrimeStore MTM lysis of the MDCK tissue culture cells.
Therefore, only a minimum 4-log TCID
50
/ml reduction was
observable.
4 L. T. Daum and others
PrimeStore MTM inactivates RNA/DNA nucleases
Nuclease digestion with RNase A, RNase T1, and
DNase was used as a simple and effective method to
examine PrimeStore protection of whole viral and
single-stranded (ss)RNA. Influenza A virus inocu-
lated in PrimeStore MTM was protected from exo-
genous addition of nucleases for 2 days at 37 xC
compared to commercial VTM, ethanol and other
lysis buffers (Fig. 1). Assuming 100 % PCR assay
efficiency, rRT–PCR cycle threshold (C
T
) differences
of 3.3 equate to an approximate tenfold change in
initial template RNA, with a C
T
of 40 representing
no detection [11]. RNA from influenza A virus
(Fig. 1a) and naked IPC ssRNA stored in Prime-
Store MTM and incubated in a pool of nucleases
(Fig. 1b) exhibited a C
T
reduction of only 5.7and1
.0,
respectively, compared to almost complete digestion
(C
T
values at or near 40) for other media after 48 h.
This equates to preservation of about 10
4
viral copies
compared to other media (Fig. 1).
PrimeStore MTM enhances RNA stability and
preservation
An influenza A(H1N1) reference strain (A/California/
04/2009) was spiked into PrimeStore MTM contain-
ing a human clinical throat swab and incubated at
25 xC (77 F) for 30 days. Influenza RNA from whole
virus was highly preserved, with an average rRT–
PCR C
T
reduction of 2.73 from day 0 baseline average
at 25 xC (Fig. 2).
Degradation of sample nucleic acids in PrimeStore
MTM can be tracked through a non-specific, IPC
(a) Influenza A virus
0
5
10
15
20
25
30
35
40
12448
Time (h)
Cycle threshold (CT)
(b) Internal positive control (IPC) ssRNA
0
5
10
15
20
25
30
35
40
124 48
Time (h)
Cycle threshold (CT)
FluA Control (–nuc)
ZR Viral RNA buffer
VTM
Ethanol
AVL lysis
PrimeStore MTM
Water + IPC Control RNA
(+nuc)
PrimeStore MTM (+ nuc)
PrimeStore MTM (–nuc)
Fig. 1. PrimeStore MTM inactivates nucleases and preserves (a) influenza A viral RNA and (b) single-stranded internal
positive control (IPC) RNA. Real-time RT–PCR analysis was performed using : 10
3
TCID
50
/ml whole influenza A(H3N2)
virus preserved in PrimeStore MTM and other media* (a), and IPC RNA (b) after incubation in RNA/DNA nucleases
(Ambion RNase A, T1, and Turbo DNase) at 37 xC for 48 h. Nucleic acid extraction at 1, 24 and 48 h was performed and
PCR amplified using PrimeMix Universal Influenza A and PrimeMix IPC assays (Longhorn Vaccines & Diagnostics). Mean
and standard error bars for duplicate extractions are shown. A C
T
reading of 40 (C
T
=40) is no amplification. [* Viral
transport media (VTM) is BD Universal Viral Transport Medium for Viruses, Chlamydiae, Mycoplasmas and Ureaplasmas
(BD, USA); AVL Lysis Buffer is Buffer AVL viral lysis buffer (Qiagen, cat. no. 1014777; USA). ZR Viral RNA Buffer is from
Zymo Research (cat. no. R1034-1-50).]
A molecular transport medium for nucleic acid testing 5
ssRNA that is included in PrimeStore MTM at an
initial concentration of 0.02 pg/ml. IPC RNA was
preserved and stable at 25 xC according to rRT–PCR
amplification using a PrimeMix IPC rRT–PCR assay
specific for the IPC RNA fragment (Fig. 2).
To demonstrate the preservation and stability of
RNA polymers in PrimeStore MTM for downstream
DNA sequencing, a stock strain (10
4
TCID
50
/ml) of
A/California/04/2009 influenza A(H1N1) 2009 was
spiked into a PrimeStore MTM and VTM and sub-
sequently incubated at 38 xC. Triplicate extractions
at days 0, 2, 7, and 14 were performed for each time-
point and subjected to standard and rRT–PCR
amplification. Traditional RT–PCR was performed
using primers to generate 574 bp and 825 bp products
from the viral segment encoding the haemagglutinin
protein (segment 4, y1795 RNA bases) (Fig. 3 a). The
574 bp amplicon from influenza A H1N1 2009 virus
preserved in PrimeStore MTM was visualized by
agarose gel electrophoresis at all time-points compared
to no amplification bands observed in extractions
from influenza virus samples stored in standard VTM
40
35
30
25
20
15
Cycle threshold (CT)
123456815192330
Time (days)
Influenza A (H1N1) virus
IPC ssRNA
Fig. 2. PrimeStore stabilizes and preserves influenza
A(H1N1) 2009 virus. A human clinical swab spiked with
5.3r10
2
TCID
50
/ml of influenza A(H1N1) A/California/04/
2009 reference virus was placed into PrimeStore MTM and
incubated at 25 xC. Triplicate samples were extracted and
analysed by real-time RT–PCR at the indicated time-point
using an assay specific for influenza A virus and the internal
positive control (IPC) single-stranded RNA piece present at
fixed concentration (i.e. 0.02 pg) within PrimeStore MTM.
Influenza H1N1-09 virus and single-stranded IPC RNA
were preserved in PrimeStore MTM for 30 days with only
minimal degradation (average 2.73 C
T
reduction) noted by
day 30 for influenza virus at 25 xC. Triplicate averages and
standard error bars are shown.
(a) Traditional RT–PCR of 574 bp and 825 bp amplicons
PS VTM PS VTM PS VTM PS VTM Pos+ Neg– Ladder
PS VTM PS VTM PS VTM PS VTM Pos+ Neg– Ladder
Day 0 Day 2 Day 7 Day 14 Controls
Day 0 Day 2 Day 7 Day 14 Controls
574 bp
825 bp
(b) Real-time RT–PCR of 194 bp amplicon
40
35
30
25
20
15
10
5
002714
Cycle threshold (C
T
)
Days at 38 °C
PrimeStore MTM
VTM
Fig. 3. RT–PCR amplification of influenza A(H1N1) 2009
stock virus in PrimeStore MTM and commercial VTM at
38 xC using: (a) standard RT–PCR with primers to amplify
574-bp and 825-bp fragments and (b) real-time RT–PCR
with primers to amplify a 194-bp fragment. A stock strain
(10
4
TCID
50
/ml) of A/California/04/2009 was placed into
PrimeStore MTM and VTM and incubated at 38 xC for 0, 2,
7 and 14 days. Traditional RT–PCR amplification of 574 bp
and 835 bp amplicons (using gel electrophoresis and UV
visualization) were observed in PrimeStore preserved
samples at days 14 and 7, respectively, compared to no
amplification in VTM-preserved samples after day 0 (a).
PrimeStore-preserved virus samples exhibited considerably
less RNA degradation (average C
T
reduction=4.0) com-
pared to no detection in VTM-preserved samples at day 14
(b). Reactions are average of triplicate with standard error
shown. Commercial VTM is Copan UTM medium for
Viruses, Chylaymida, Mycoplasma & Ureaplasma (Copan
Diagnostics, Italy).
6 L. T. Daum and others
after day 0 (Fig. 3 a). The PCR band corresponding to
the 825 bp amplification was evident to day 7 in virus
stored in PrimeStore MTM
TM
,comparedtonoam-
plification in virus stored in VTM after day 0 (Fig. 3a).
Similar results were observed using rRT–PCR
for amplification of a 194-bp fragment specific for a
conserved region of influenza A Matrix gene. Influ-
enza virus preserved in PrimeStore showed consider-
ably less degradation according to rRT–PCR (average
C
T
reduction over 14 days=4.0) compared to no de-
tection observed in virus stored in VTM (Fig. 3 b).
PrimeStore is compatible with commercial silica- and
bead-based extraction kits
Commercial extraction kits are available in a variety
of formats with most exploiting the basic principle of
nucleic acid affinity for glass silicon dioxide particles
present on filters or beads. To demonstrate Prime-
Store MTM compatibility with commercial kits, refer-
ence influenza A(H1N1) virus (A/California/04/2009)
was inoculated into PrimeStore containing a human
throat swab specimen at two concentrations, 10
3
TCID
50
/ml and 10
1
TCID
50
/ml, and extracted using
two commercial spin-column and two bead-based kits
(Fig. 4). Viral influenza RNA in PrimeStore MTM
was detected by rRT–PCR after extraction in all four
commercial kits at high (10
3
TCID
50
/ml) and low (10
1
TCID
50
/ml) viral loads. Additionally, the IPC RNA
(0.02 pg/ml) present in PrimeStore MTM was also
detected by rRT–PCR after extraction using all four
kits at levels consistent with the manufacturer’s rec-
ommendations [12].
DISCUSSION
Several companies such as Becton Dickinson, and
Copan offer viral collection media for preserving
viable organism for culture analysis. Unknown speci-
mens collected in these media remain viable and are
typically transported frozen to the laboratory and
treated as biohazardous and potentially infectious.
Therefore, there is considerable expense and risk of
infection associated with the collection and transport
of clinical specimens to reference laboratories.
Krafft et al. [13] evaluated the use of respiratory
samples fixed in ethanol as an alternative to com-
mercial transport media using rRT–PCR. While
ethanol may provide a suitable medium for influenza
and other common upper respiratory pathogens,
its use presents several other concerns including
alcohol-shipping restrictions in many countries, high
flammability, and the potential for incomplete lyses of
certain microbes.
In a study by Blow et al. [14], viral RNA from field-
collected arboviruses was stabilized at 32 xC for 48 h
using the AVL lysis buffer from a commercially
available Qiagen extraction kit. While the samples are
lysed and presumably non-infectious, the short, 2-day
window of RNA preservation in Qiagen AVL makes
this method unrealistic, even for routine pathogen
surveillance.
The use of RNALater
TM
(Ambion, USA) has been
reported in the literature as a solution for the preser-
vation of collected specimens for molecular analysis
[15, 16]. RNALater is a commercially available pro-
duct designed for the preservation and maintenance
of whole tissues for histology and microscopy of cells
and microbes [17], but its use has also included the
collection of field specimens for molecular analysis
[18–20]. RNALater contains a high concentration
of ammonium sulfate for maintaining phospholipid
integrity and intact cells. Several reports indicate
that tissues and viruses from samples preserved in
RNALater remain viable for extended periods of
time and at a variety of temperatures [21, 22].
40
35
30
25
20
15
10
5
0103 TCID50/ml
Cycle threshold (CT)
101 TCID50/ml ssRNA IPC
Ambion RNAqueous-Micro
Qiagen Viral Mini
Invitrogen ChargeSwitch
Ambion MagMax
Fig. 4. PrimeStore molecular transport solution is compat-
ible with commercial bench-top nucleic acid extraction kits
including silica- and bead-based varieties. Two concen-
trations (10
3
TCID
50
/ml and 10
1
TCID
50
/ml), representing
clinically relevant viral loads, i.e. a high and low, of stock
H1N1 virus (A/California/04/2009) were inoculated into
PrimeStore containing a clinical throat swab. Extractions
were performed using two common silica- and bead-based
nucleic acid kits (according to manufacturer’s recommend-
ations) and subjected to real-time RT–PCR analysis using
an ABI 7500. Mean and standard error for triplicate ex-
tractions are shown.
A molecular transport medium for nucleic acid testing 7
Maintaining viability of viruses and other microbes
can be dangerous for many groups interested in safe,
non-infectious transport of collected specimens for
molecular-based diagnostics.
PrimeStore MTM was developed specifically to
overcome these limitations in clinically collected
specimens earmarked for routine downstream mol-
ecular diagnostics. Since PrimeStore MTM inacti-
vates and kills a wide range of microbes (Table 1) it is
well-suited for field collection in remote areas, triage
centres, border crossings and during pandemics where
cold-chain, transport and dissemination of potentially
infectious pathogens are a concern. PrimeStore MTM
lyses and inactivates microbial pathogens so they are
non-viable; thus an alternative transport media is re-
quired for traditional culture. Preliminary data from a
study in progress has demonstrated that PrimeStore
MTM rapidly kills M. tuberculosis from clinical
sputum samples (Dr Nazir Ahmed Ismail, Medical
Microbiologist, University of Pretoria and NHLS,
Pretoria, South Africa, personal communication).
PrimeStore MTM is an ambient thermostable, pro-
prietary molecular transport medium that rapidly
inactivates nucleases (Fig. 1) and stabilizes and pre-
serves released nucleic acids including labile RNA
according to rRT–PCR analysis (Fig. 2). Further-
more, PrimeStore MTM stabilizes and preserves
RNA polymers from hydrolysis and oxidation, and
promotes thermostability at temperatures as high as
37 xC for at least 14 days (Fig. 3).
A unique IPC ssRNA that is pre-mixed (3r10
5
target copies/ml) into PrimeStore MTM (and is thus
stabilized) can be amplified by rRT–PCR to verify
sample stability from the time of sample collection
through extraction and detection (Fig. 2). The IPC
RNA serves as a carrier species for low-level samples,
controls for nucleic acid extraction and monitors
sample integrity from the point of collection to de-
tection. The RNA IPC in PrimeStore MTM is a syn-
thetically produced (in vitro) 114-bp ssRNA polymer
that is non-homologous by BLAST analysis to com-
mon upper respiratory pathogens and normal flora. A
developed rRT–PCR assay (PrimeMix IPC, Longhorn
Vaccines & Diagnostics) that targets this synthetic
IPC RNA polymer is used as a uniplex rRT–PCR
assay. A clinical collection medium containing exo-
genous IPC RNA fragments is important for ensuring
integrity of transported or stored specimens for a wide
variety of molecular-based detection systems.
PrimeStore MTM will also facilitate standard
sequencing and meta-genomic analysis of original
clinical samples by improving the quality of microbial
nucleic acids in original specimens when they finally
arrive in the laboratory. Recovery of RT–PCR ampli-
fication fragments over 1400 bases (data not shown)
has been observed from viral RNA preserved and
shipped in PrimeStore MTM at ambient temperature
for several weeks. In harsh conditions, i.e. 38 xC
incubation, RT–PCR amplification of 574-bp and
825-bp fragments were observed from PrimeStore
preserved virus where no amplification was observed
from stock virus in commercial VTM (Fig. 3 a).
The primers used to generate the 825-bp fragment
represent the largest haemagglutinin gene fragment
described by the WHO for influenza A(H1N1) se-
quencing [10]. Ambient temperature collection and
preservation of large RNA fragments from clinical
samples in PrimeStore MTM may be highly useful to
groups who wish to perform direct DNA sequencing,
genotyping, meta-genomic analysis and other NAT
directly from original specimens.
In a 2007–2008 prospective paediatric clinical
study, nasal washings preserved in PrimeStore
MTM were subsequently analysed for influenza
by rRT–PCR [23] and DNA sequencing of viral
surface genes (Daum et al., unpublished ob-
servations). During the 2009 swine pandemic cli-
nical throat swabs preserved in PrimeStore MTM
were transported at ambient temperature and sub-
jected to entire genome sequencing of swine influenza
A(H1N1) virus using a Roche 454 system (Haung
et al., unpublished data). In this work Haung
et al. characterized four complete influenza genomes
from the Dominican Republic, Columbia, Nicaragua,
and Russia from swabs in PrimeStore MTM
[GenBank accession numbers CY0Y3099-CY0Y3106,
CY049833-CY049839, CY044152-CY044159, CY049896-
CY049903; Haung et al., unpublished data].
PrimeStore MTM is compatible with many com-
mercial extraction kits (Fig. 4). Nucleic acids are ex-
tracted directly from PrimeStore MTM according to
standard manufacturer’s protocol with only minor
differences noted in C
T
values between column- and
bead-based kits. Previous studies have shown that
spin-column kits are slightly more robust compared
to bead-based kits with certain sample types [24, 25],
but bead-based kits may be better when certain
sample matrices, i.e. faecal and environmental are
analysed. Additional studies are currently underway
to evaluate the utility of PrimeStore MTM for use
with other samples matrices such as faeces, blood,
environmental samples and veterinary specimens.
8 L. T. Daum and others
A 2010 influenza study that compared swabs in liquid
Stuart’s transport media (RTLS) and PrimeStore
MTM from pigs experimentally infected with H1N1
influenza A virus found that virus from swabs in
PrimeStore MTM and not Stuart’s medium was de-
tected at 5 days post-infection, a time when shedding
is typically decreased or non-detectable [26].
While PrimeStore MTM effectively lyses microbal
pathogens, it is more than simply a lysis buffer as
evident by comparison to typical lysis buffers from
commercial extraction kits (Fig. 1). PrimeStore MTM
is a blend of eight reagents that have been optimized
to enhance RNA/DNA preservation after cellular
lysis. PrimeStore MTM does not exhibit inhibition
during nucleic acid extraction and PCR testing
and has been shown to improve optimal recovery of
influenza RNA (Fig. 3).
Notable reductions (4–5 C
T
s) in initial rRT–PCR
C
T
values are routinely observed from virus in-
oculated in commercial VTM compared to equal
amounts of virus in PrimeStore MTM (Fig. 3 b). This
suggests that extraction and/or PCR inhibitors are
present in commercial VTM that effect RNA detec-
tion during rRT–PCR. This may be problematical
for groups who routinely extract RNA from original
clinical samples collected in commercial VTMs for
rRT–PCR or gene sequencing analysis. Accordingly,
there is a need for an effective clinical VTM that
serves the dual role of ensuring the viability of col-
lected microorganisms for subsequent culture and
propagation procedures and does not substantially
interfere with downstream NAT processes.
In February 2010, PrimeStore MTM received
FDA-Emergency Use Authorization as part of the
complete Longhorn Influenza A/H1N1-09 Prime
RRT–PCR Assay
TM
[12]. PrimeStore is the first
molecular transport medium to receive EUA FDA
approval, and the first to contain an IPC to con-
trol for specimen degradation from collection to
detection.
ACKNOWLEDGEMENTS
We acknowledge the countless hours of effort
from scientists, physicians and students alike who
contributed to the development and clinical utility
of PrimeStore MTM in molecular diagnostics.
Specifically, Deena E. Sutter, M.D., Marty Ottolini,
M.D., Aryeneesh K. Dotiwala, John Rodriquez,
Demetra Kelenis, Jeff Fischer and Lou Schriefer.
DECLARATION OF INTEREST
Drs Luke T. Daum and Gerald W. Fischer and
Ms. Susan A. Worthy are employees of Longhorn
Vaccines & Diagnostics.
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