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JCM Accepts, published online ahead of print on 22 January 2014
J. Clin. Microbiol. doi:10.1128/JCM.03453-13
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Comparative Assessment of Automated Nucleic Acid Sample Extraction Equipment for
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Biothreat Agents
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Warren Vincent Kalinaa, Christina Elizabeth Douglasa, Susan Rajnik Coynea,
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Timothy Devin Minoguea#
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Diagnostic Systems Division, United States Army Medical Research Institute of Infectious
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Disease, Fort Detrick, MD 21702a
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Running Head: Agent Extraction Equipment Comparison
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#Address correspondence to Timothy D. Minogue, [email protected]
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Abstract:
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Magnetic beads offer superior impurity removal and nucleic acid selection over older extraction
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methods.
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extraction performance of biothreat agents in blood or buffer was evaluated. All instruments
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showed excellent performance in blood; however, the easyMAG® had best precision and
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versatility.
The easyMAG®, MagNA Pure, EZ1 Advanced-XL, and Arrows’ nucleic acid
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Keywords: Magnetic separation, Bacillus anthracis spores, Y. pestis, Vaccinia Virus, and
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Venezuelan Equine Encephalitis Virus
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In the early days of molecular biology, nucleic acids were extracted from large laboratory
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grown agent preparations that yielded sparse product [1]. After the discovery of DNAses,
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RNAses, and chaotropic salts, purification procedures were generated that yielded consistent,
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measurable nucleic acid product [2-4]. Several current clinical diagnostic extraction procedures
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separate nucleic acid by magnetic beads which harmonizes older methods with user-friendly and
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precise techniques [4, 5]. Such methods have become automated whereby extraction instruments
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use magnets and silica-coated magnetic beads to bind nucleic acid through electrostatic
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interactions while impurities are washed away with buffers [6, 7].
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Numerous high-throughput, magnetic separation, extraction technologies offer maximal
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recovery, purity, and minimal operator involvement but use different beads, protocols, or
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reagents. Material and process disparities potentially affect agent extraction efficiency in
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specific matrices [8-10]. Few studies have compared the performance of automated nucleic acid
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extraction platforms using clinical specimens that contain biothreat agents [10, 11]. These facts
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warranted an analysis of instrument effects on agent-specific detection by real-time PCR.
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Therefore, extraction performance was evaluated for Bacillus anthracis, Yesinia pestis, Vaccinia,
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and Venezuelan Equine Encephalitis (VEE) virus in blood and buffer on the easyMAG®
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(bioMérieux, Durham, NC), MagNA Pure Compact (Roche Diagnostics, Indianapolis, IN), EZ1
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Advanced XL (Qiagen, Valencia, CA), and Nordiag Arrow (Autogen, Holliston, MA).
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Yersinia pestis (Colorado92) (ANG-YERS078), VEE 1A/B Trinidad Donkey Strain
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(ANG-VEETRIN), Vaccina Lister (ANG-VAVIEL), and Bacillus anthracis Ames (ANG-
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BACI008) were obtained inactivated from the U.S. Department of Defense Critical Reagent
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Program (Fort Detrick, MD)[12]. Inactivation was previously shown to not affect agent-specific
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real-time PCR assays and other extraction procedures [13]. All agents were serially diluted 8
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times from titers (CFU or PFU/mL) of 1×105 to 7.8×102. Eight replicates were prepared for each
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dilution in either Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies, Grand
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Island, NY) or single donor EDTA whole blood (Bioreclamation, Westbury, NY) which was
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duplicated for each instrument with all samples diluted on the same day prior to storage at -80°C.
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Varying instrument throughputs necessitated sample arrangement with minimal 2 dilution
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replicates per run. Agents were extracted from blood and buffer with kits or settings
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recommended by the applicable instrument’s vendor (Table 1).
Dilution linearity, precision, instrument effects on limit of detection (LOD) and matrix
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were measured by real-time PCR after extraction. Intra and inter-assay precision, defined as
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variation between replicates within a run and between separate runs respectively, were calculated
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as %CV. Primers for B. anthracis PA, Y. pestis PIM, VEEV nsp4, and Vaccinia E9L genes were
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used in real-time PCR assays as previously described on a Lightcycler 480 (Roche Diagnostics,
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Indianapolis, IN) [13, 14]. Real-time PCR Cq values between instruments and matrices were
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evaluated by t-tests, one-way-analysis-of-variance, and probit regression in GraphPad Prism®
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[15].
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Inter and intra-assay precision was mostly within tolerance for all instruments except
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Arrow-extracted buffer samples (Table 2). For all agents in blood and buffer, the easyMAG®
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had the best overall precision followed by the MagNA Pure, whose B. anthracis inter-assay
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extraction precision in buffer was appreciably lower. Similarly, the EZ1’s intra and inter-assay
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precision was good for all agents except Vaccinia in buffer/blood. Lastly, the Arrow failed to
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extract all agents in buffer, but showed good precision, excluding Vaccinia, for all agents in
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blood. Incidentally, the easyMAG® uses guanidinium thiocyanate (GuSCN) in lysis and
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washing steps, and the Arrow and EZ1 use guanidinium hydrochloride (GuHCl) during lysis,
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binding, and washing steps. Comparatively, GuSCN is a stronger chaotropic salt and denaturant
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than GuHCl that superlatively inactivates nucleases, opens cells to release nucleic acid, induces
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salt bridge formation, and disrupts H-bonding and subsequently may have resulted in enhanced
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precision shown here [4].
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There was no significant difference in Cq values between instrument sample dilution
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curves, and each agent failed to produce linear dilution treadlines in blood or buffer (r^2<0.95)
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(data not shown). Because samples were made in one batch, poor linearity was extraction
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instrument dependent.
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Statistical analysis of precision and linearity provided definitive metrics for adjudicating
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instrument performance; however, detection of organism in a sample for incorporation into the
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diagnostic pipeline required further consideration, i.e. effects on real-time PCR LOD. Similar to
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precision and linearity analysis, real-time PCR confirmed better LODs for easyMAG® extracted
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B. anthracis at 840 CFU in blood whereas the Arrow, MagNA Pure, and EZ1 yielded lower
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quantities of B. anthracis nucleic acid in blood, and all instruments extracted less material in
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buffer (Table 2). Yields for Y. pestis, Vaccinia, and VEE were more equivocal than B. anthracis
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in both matrices (results not shown). Considering all agents, the easyMAG®, MagNA Pure,
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and EZ1 extracted significantly different amounts of nucleic acid between buffer and blood
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except VEE on the EZ1 (Figure 1). Incidentally, nucleic acid released from enveloped viruses
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doesn’t require harsh denaturants, but peptidoglycan and endospores, which are recalcitrant to
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lysis even in NucliSENS lysis buffer, may account for observational differences between
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instruments[16].
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Intuitively blood, teeming with inhibitors, was expected to reduce extraction yields;
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however, this wasn’t observed thus suggesting GuSCN or GuHCl concentrations in extraction
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buffers sufficiently inactivate nucleases. Samples in DPBS, extracted with the same reagents
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used for blood, may be exposed to unnecessary high concentrations of harsh, ionic chaotropic
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salts that affect silica binding and nucleic acid integrity. Concerning the Arrow’s performance in
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buffer, chemistry and mechanics may interplay. Buffer sample volumes were observed as not
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being pipetted accurately by the instrument, thus insinuating liquid retention capacity is
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calibrated for viscous and not for non-viscous solutions. Of note, our laboratory has used
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magnetic bead extraction technology to extract the same live and killed agents used in this study
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without any difference in results (unpublished observation) and therefore had no profound effect
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on results presented here.
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Overall, the easyMAG® had superior precision and extraction yields for most agents
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with best throughput. However, the EZ1 extracted VEE equivocally regardless of matrix but had
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lower precision. All instruments extracted material in blood with varying degrees of
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performance, which was the primary focus of this study. Since the EZ1 has greater versatility for
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viral samples in different matrices, reasonably good throughput (14 samples/run), and small
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footprint, such an instrument may be advantageous in clinical labs that frequently process viral
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samples; however, precision, matrix robustness, throughput, typical agent type, and extraction
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time should be major instrument considerations.
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Acknowledgments:
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This work was supported by the Joint Science & Technology Office for Chemical & Biological
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Defense under Grant No. CB3901. Opinions, interpretations, conclusions, and recommendation
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are those of the author and are not necessarily endorsed by the U.S. Army.
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References:
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Nucleic Acid Extraction Methods with manual Extraction. Journal of Molecular
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Nabuurs-Franssen MH, de Vries MC, Horrevorts AM, Klaassen CHW. 2010.
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Interlaboratory Evaluation of Different Extraction and Real-Time PCR Methods for
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Detection of Coxiella burnetii DNA in Serum. Journal of Clinical Microbiology. 48(11):
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Food Microbiology. 150(2-3): p. 122-127.
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Figure 1. Percentage of extracted agent dilutions (1×105 to 7.8×102) that were statistically
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different between blood and buffer for each instrument. The Arrow is not shown because
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therewas no data for buffer to make comparisons. With the exception of the EZ1 for VEEV, the
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easyMAG® had similar but overall fewer instances of significantly different data between blood
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and buffer samples.
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Table 1. Instrument extraction parameters
Method
(company)
Kit
Binding Lysis
Buffer
NucliSens
easyMAG®
bioMérieux
NucliSens
magnetic kit
NucliSENS
Lysis Buffer
(GuSCN)
EZ1 XL
Qiagen
EZ1 Virus
Mini Kit v2.0
Buffer AL
(GuHCl)
MagNA Pure
Compact
Roche
Arrow
NorDiag
Nucleic Acid
Kit I
GuSCN
Unknown
Lysisblood/buffer
Blood DNA
200
30% GuHCl
30% Perchlorate
50% Ethanol
10 mM TrisHCl pH8.0
None
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Elution
Buffer Used
NucliSENS
Extraction
Buffer 3
+heat
Buffer AVE
All instruments required sample preparation to be external
External
Step1
Lysis-blood
None
Table 2. Performance Results
Instrument/Kit
Matrix
Intra-Best1
Intra-Worst
easyMAG®
Buffer
Blood
YP,VV
NA
BA
NA
MagNA Pure
Buffer
Blood
Buffer
Blood
Buffer
YP, VEEV
BA, YP, VEEV
BA, YP, VEEV
BA, YP, VEEV
None6
Blood
BA, YP, VEEV
BA
VV
VV
VV
BA, YP, VV,
VEEV
VV
EZ1-Advanced XL
Arrow
%Pass2
Intra
96.6
100
%Pass3
Inter
100
92.9
BA LOD4
1488
840
97.1
99.1
98.7
95.6
55.0
82.1
96.7
76.7
79.5
0.00
4744
2771
10719
8628
ND5
94.4
87.4
1381
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Agent(s) (YP=Y. pestis, BA=B. anthracis, VV=Vaccina Virus, VEEV=Venqualan Equine
Encephalitis Virus) with lowest numbers of intra-assay replicates with %CV<5. Several
individual agents had the same pass rate and are shown jointly.
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Percentage of replicates where intra-assay precision %CV<5 for all agents combined
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Percentage of replicates where inter-assay precision %CV<2 for all agents combined
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Expressed as estimated CFU
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Not Determined
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For most agents failure exceeded 50%