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Short-term Stability of Pathogen Specific Nucleic Acid Targets in Clinical Samples 1
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Mohammad R. Hasan1,2*, Rusung Tan1,2, Ghada N. Al-Rawahi1,2, Eva Thomas1,2, Peter Tilley1,2 3
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1Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's 5
and Women's Health Centre of BC, Vancouver, BC, Canada and 2Department of Pathology and 6
Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 7
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Running title: Stability of Pathogen Specific Amplification Targets 9
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Keywords: Pathogen, Nucleic acid amplification tests (NAATs), nucleic acid stability, clinical 11
specimens, storage temperature 12
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*Corresponding author: 15
Mohammad R. Hasan, Microbiology Laboratory, BC Children's & Women's Hospitals, Rm 16
2F59A, 4500 Oak Street, Vancouver, BC V6H 3N1, Canada 17
Phone: (604)-875-2345 Ext. 5283 18
FAX: (604)-875-3777 19
E-mail: Rubayet.Hasan@cw.bc.ca 20
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.02659-12 JCM Accepts, published online ahead of print on 10 October 2012
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Abstract 24
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The stability of pathogen specific DNA or RNA amplification targets in clinical samples 26
following short-term storage at room temperature, 4oC and -80oC was assessed by real time PCR. 27
In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, 28
irrespective of storage temperature. In unextracted samples, temperature-dependent loss of 29
targets (p<0.05) was observed in serum and cerebrospinal fluid specimens while no changes 30
were observed for EDTA blood samples. 31
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Nucleic acid amplification tests (NAATs) to detect infectious pathogens are now widely used in 47
clinical microbiology laboratories. Although these assays provide high sensitivity, specificity, 48
and rapid turn-around time, special laboratory practice and procedures are required to avoid false 49
positive and false negative results (1,2). Optimal application of NAATs depends on a number of 50
pre-analytical factors such as specimen collection, transport and storage conditions (3,4). These 51
factors impact the stability of pathogen-associated nucleic acids (NA) which must be protected 52
from physical, chemical or enzymatic degradation during specimen transport and handling. The 53
most important threats to the stability of NA targets in clinical samples are nucleases, such as 54
DNases and RNases, which may be released from lysed human cells and microorganisms (5). In 55
real life clinical situations, questions arise over whether clinical samples or NA extracts, left at 56
room temperature (RT) for a few days or stored in the refrigerator for a week are still valid for 57
molecular testing. Although there are published guidelines for the collection, transport and 58
storage of specimens for molecular tests, described in CLSI (4), they are mostly based on 59
opinions and limited experimental data. Published data on the stability of pathogen-specific 60
amplification targets are also limited to individual pathogens and/or specimen types (3,4,6-10, 61
12). Therefore, in this study, we undertook a broader study to compare the stability of extracted 62
and unextracted clinical samples over time and at different temperatures. 63
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To determine the short-term storage stability of purified DNA and RNA, NA was extracted from 65
known positive specimens containing the following: Neisseria meningitidis in EDTA blood; 66
cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in serum; and influenza A virus in 67
nasopharyngeal washes (NPW). All samples were extracted using the QIAsymphony 68
virus/bacteria kit in an automated DNA extraction platform QIAsymphony SP (Qiagen) except 69
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for EDTA blood, which was extracted manually using QIAamp DNA Blood Mini Kit (Qiagen). 70
Purified eluates were stored in aliquots under sterile conditions at room temperature (RT) (22-71
25oC), 4oC and -80oC for 0-30 days. 72
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For unextracted samples, the target pathogen and the specimen types were chosen so that they (i) 74
represent the most common specimen type for the respective pathogen and ii) had not previously 75
been studied. EDTA blood, serum, cerebrospinal fluid (CSF) and NPW samples were spiked 76
with a N. meningitidis B strain (ATCC 13090), a CMV patient isolate, a human enterovirus 70 77
strain (ATCC VR-836) and an influenza A virus patient isolate, respectively, within 4 hours of 78
collection (Supplemental methods). NA from spiked samples was extracted in the same way 79
following storage at RT, 4oC and -80oC for 0-16 days. Extracted nucleic acids were analyzed 80
either by in-house (Supplemental method and Table 1) or commercial, probe-based, real time 81
PCR assays. Human enterovirus RNA was detected using a commercial eQ-PCR™ EnV 82
Detection Kit (Trimgen Corporation). For kinetic analysis, CT values for each pathogen were 83
plotted against the number of days the samples were stored at different temperatures. The half-84
lives of pathogen-associated nucleic acid targets were obtained from the linear equation 85
(Supplemental methods). For average analysis, fold changes in the relative quantity of 86
amplification targets were calculated based on CT values using the equation FC=2^(-dCT), where 87
dCT=(CT,day X- CT,day 0) (11). The percent loss of targets was calculated from fold changes from 88
the quantity of targets at day ‘0’. Statistical significance was calculated by paired and 2-tailed, 89
student’s T-test and p<0.05 was considered to be statistically significant. Statistical differences 90
between multiple experimental groups were calculated by ANOVA followed by post-test using 91
Bonferroni correction at 95% confidence interval. 92
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There was no loss of extracted N. meningitidis, CMV and EBV DNA derived from EDTA blood 93
and serum samples after storage at RT, 4oC and -80oC for 0, 3, 7, 14 and 30 days (Fig. 1A-i,C-94
i,D-i). To account for sample to sample variation, we performed further tests on additional 95
samples at days 0 and 30 of storage at different temperature, and found no significant differences 96
in the relative quantity of specific amplification targets (Fig. 1A-ii, C-ii, D-ii). In general, DNA, 97
when purified, and stored in a preferred buffer such as Tris/EDTA (pH 7.2) is considered to be 98
stable for at least one year at 2-8oC, and for longer terms at -70oC or lower (3). However, it is 99
important to note that the DNA purity could be dependent on the specimen type and the 100
extraction methods, which could subsequently influence the stability of purified DNA. Our 101
findings suggest that DNA extracted from EDTA blood or serum samples, using an automated 102
and magnetic bead-based extraction method, may be safely stored at RT or lower without any 103
loss for up to one month. Unlike the case with DNA, it is recommended that purified RNA be 104
stored at -70oC or lower, and at a slightly alkaline pH (7.1-7.5) (3). In our lab setting, we 105
observed a small (~1 CT; p<0.05) loss of influenza A RNA, which was not temperature 106
dependent (between RT to -80oC), in purified NPW extracts following storage for a period of one 107
month (Fig. 1B). These results indicate that temporary storage of extracted nucleic acids from 108
clinical samples at RT or 4oC is unlikely to affect NAAT results. 109
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Unlike extracted samples, where purified DNA and RNA molecules are maintained in a defined 111
buffer, crude clinical samples represent a complex biological environment, where an interplay of 112
degradative proteases and nucleases may determine the fate of pathogen-associated amplification 113
targets. Previously published observations on the stability of microbial DNA in clinical samples 114
include i) Trichomonas vaginalis DNA, which was stable for 1 day at RT and 3 days at 4oC in 115
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urine samples (6) ii) spiked Treponema pallidum DNA in CSF which was detectable after 96h of 116
storage at RT and 4oC (12) iii) HSV DNA in positive clinical specimens remained stable for at 117
least 16 months at -20°C (7), and iv) HBV DNA in positive plasma samples was stable at 25oC 118
and at 5oC for at least 25 days (8), whereas, in serum they were stable only for 5 days at 4oC and 119
-70oC and less stable at RT (10). Our results on the stability of spiked N. meningitidis DNA in 120
EDTA blood, and CMV DNA in serum and EDTA blood samples correlate well and 121
complement these observations. Whereas both N. meningitidis and CMV DNA were highly 122
stable in EDTA blood for two weeks at any storage temperature used, significant loss (p<0.05 by 123
ANOVA and post-test) of CMV DNA was observed in serum samples in a temperature-124
dependent manner (Fig. 2A-B). The half-life of CMV DNA based on PCR assays was <1, 2, and 125
3 days when stored at RT, 4oC and -80oC, respectively (Table 1). 126
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Although a loss of RNA targets in crude samples is expected during storage, unless frozen, we 128
noted no dramatic, logarithmic decrease in human enterovirus and influenza A RNA levels in 129
CSF and NPW samples, respectively after two weeks. A small but statistically significant 130
reduction of enterovirus RNA (65%; p<0.05) at RT, and influenza RNA (>50%; p<0.001) at all 131
temperatures was observed (Fig. 2C-D). The half-life of enterovirus RNA in CSF samples at RT 132
was 9 days (Table 1), which correlates well with that of HIV-1 RNA (7 days) in plasma samples, 133
as previously reported (8). Similarly, at 4oC, enterovirus RNA maintained its integrity for 134
approximately two weeks in this study as seen in a previous HIV-1 study (8). The results of 135
influenza A RNA in NPW samples were somewhat surprising as no temperature-dependent 136
effects were detected. One limitation of this study is that we did not analyze RNA stability in 137
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EDTA blood samples. However, a previous study found HCV RNA stable in EDTA blood for up 138
to 96 hours (9), similar to what we observed for DNA targets. 139
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In conclusion, we observed minimal loss of pathogen NAAT targets for both extracted and 141
unextracted clinical samples within a few days of specimen collection. None of the NAAT 142
targets became undetectable during the study period, which was limited to 1 month and 2 weeks, 143
for pure extracts and crude samples, respectively, to represent most common scenarios in a 144
diagnostic microbiology laboratory. The temperature-dependent loss of targets in serum and CSF 145
samples indicates possible enzymatic degradation. In contrast, EDTA blood samples were found 146
to be the most protective medium for nucleic acid stability. This may be because EDTA 147
sequesters metal ions required for enzymatic activity and thus inhibits nucleases and proteases. 148
Based on these observations, we conclude that, if a processing delay is unavoidable, EDTA 149
blood or plasma samples are preferable to serum samples for real time PCR detection of bacterial 150
or viral pathogens. 151
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tubes on hepatitis C virus level in whole blood samples. J. Clin. Microbiol.39(5):1788-183
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Figure legends 206
FIG 1: Storage stability of DNA and RNA in post-extracted clinical samples. Nucleic acid 207
extracts from clinical samples known positive for N. meningitidis (A), Influenza A virus (B), 208
CMV (C) and EBV (D) were assessed for respective targets by Taqman PCR at day ‘0’ and 209
following storage at RT, 4oC and -80oC for different time intervals as indicated. A-D(i) Kinetic 210
data based on CT values plotted against number of days stored at different temperature. A-D(ii) 211
Average of fold changes calculated from CT values, with respect to CT values at day ‘0’. Baseline 212
CT values in different samples ranged from 29.9 to 38.5 for N. meningitidis, 28.4 to 39.5 for 213
CMV, 28.2 to 38.5 for EBV and 21.3 to 34.8 for Influenza A virus. The CT value difference 214
because of intra-assay variation between duplicate runs was <1 CT. Error bars = SEM; statistical 215
significance calculated by pair-wise student’s T-test (2-tailed); *p<0.05, **p<0.01, ***p<0.001. 216
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FIG 2: Storage stability of DNA and RNA in pre-extracted clinical samples. Clinical 218
samples were spiked with N. meningitidis (A), CMV (B), Enterovirus (C) and Influenza A virus 219
(D). Nucleic acid extraction and Taqman PCR for the respective targets were performed on 220
aliquots of samples at day ‘0’ and following storage at RT, 4oC and -80oC for different time 221
intervals as indicated. A-D(i) Kinetic data based on CT values plotted against number of days 222
stored at different temperature. A-D(ii) and B(iii) Average of fold changes calculated from CT 223
values, with respect to CT values at day ‘0’. Baseline CT values in different spiked samples 224
ranged from 31.9 to 34.0 for N. meningitidis, 29.6 to 31.7 for CMV, 32.7 to 34.1 for enterovirus, 225
and 33.9 to 37.1 for Influenza A. The CT value difference because of intra-assay variation 226
between duplicate runs was <1 CT. Error bars = SEM; statistical significance compared to data at 227
day 0 calculated by pair-wise student’s T-test (2-tailed); *p<0.05, ***p<0.001. Multiple group 228
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comparisons for CMV DNA levels in serum samples were made by ANOVA and post-test (95% 229
confidence interval) indicating a statistically significant (p<0.05) difference between different 230
groups. 231
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Table 1: Half life and % loss of pathogen specific amplification targets in different types of
clinical specimens.
Pathogen Specimen type Days stored
Half life (days) % loss of target after 2 weeks
RT 4oC -80oC RT 4oC -80oC
N.meningitidis EDTA Blood 16 - - - No loss No loss No loss
CMV Serum 16 <1 2 3 79.7±5 50±10 36.9±12
EDTA Blood 16 - - - No loss No loss No loss
Human Enterovirus CSF 16 9 - - 82.1±9 No loss No loss
Influenza A Nasopharyngeal aspirates
14 6 6 10 74±23 70±25 66±27
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