MagNA Pure System Application Note No. 1
Performance Efficient Extraction
of Viral DNA and RNA from a
Variety of Patient Sample
Materials in Routine Diagnostics
using a Single Standardized Protocol
with the MagNA Pure 96 System
René Minnaar and Richard Molenkamp
Department of Medical Microbiology,
Academic Medical Center, (AMC),
Amsterdam, The Netherlands
June 2011
1 Abstract
In our molecular diagnostic laboratory, we routinely use the
MagNA Pure 96 System for nucleic acid extraction. In this
paper, we show that the MagNA Pure 96 System is reliable
and convenient for the extraction of viral RNA and/or DNA
from a variety of patient sample materials.
Extraction was very sensitive and efficient, resulting in the
detection of 10 – 100 viral copies, depending on sample and
virus type. Effects resulting from PCR inhibition were
excluded by experiments using Internal Controls.
Furthermore, we evaluated one single extraction protocol
and one single RT-PCR protocol that can be used generically
for all sample and virus types in the same run.
Detection using the Roche LightCycler ® 480 Real-Time
PCR Instrument revealed that extraction from all 6 materials
tested was very efficient. Even with known difficult
materials, such as faeces and biopsies, the MagNA Pure 96
System performed well.
In addition, we extracted nucleic acid from 2 different
volume inputs (50 and 200 µl) of EDTA whole blood from
patients positive for CMV. Higher inputs led to higher
sensitivity in 7/8 samples, crossing point (Cp) cycle values
were approximately 1.7 cycle lower while Cp values for IC
remained similar.
For life science research only.
Not for use in diagnostic procedures.
2 Introduction
The Academic Medical Center (AMC) is one of the eight
university medical centers in the Netherlands. The three
principal tasks are patient care, research and medical
education. The AMC has over 1,000 beds and more than
50,000 clinical and daycare admissions. The Department
of Medical Microbiology participates in all three principal
tasks of the AMC. Patient care is provided by laboratory
diagnosis and consultation. Within the department of
medical microbiology, the molecular diagnostic unit (MDU)
provides molecular detection of a broad range of
microorganisms.
Over the last decade, the contribution of molecular
techniques to the diagnosis of infectious disease in medical
microbiology laboratories has increased significantly.
Not only the amount of samples processed and the choice
of targets has increased, also the nature and variety of
the patient materials tested has grown. Development of
in-house or home-brew molecular diagnostic tests require
efficient and convenient nucleic acid extraction protocols.
Due to the increased size of sample flow, nucleic acid
extraction is preferably automated to process many samples,
in as little time as possible.
For this, there is a need for a fast, automated high-throughput
purification system that can handle a variety of materials,
preferably in the same extraction run and using the same
extraction protocol. To investigate if the MagNA Pure 96
System is able to efficiently extract nucleic acid from various
patient materials, we spiked different clinical materials
with various amounts of different characterized virus stocks.
Internal controls were added to all samples to monitor
extraction efficiency of the MagNA Pure 96 System. The
readout was performed using real-time PCR with hydrolysis
probes in a Roche LightCycler ® 480 Real Time PCR
Instrument.
3 Materials and Methods
Virus stocks and patient material
DNA viruses Human Adenovirus (HAV) and Cyto­megalo­
virus (CMV), as well as RNA viruses Human Parechovirus
(HPeV) and Influenza A virus, were isolated and cultured
from patient sample materials sent to the laboratory for
molecular diagnosis. Virus stocks were TCID50* quantified.
* M edian tissue culture infective dose; the amount of a cytopathogenic
agent (virus) that will produce a cytopathic effect in 50% of cell ­cultures
inoculated, e xpressed as TCID 50/ml.
Table 1: Treatment of various patient sample materials to be extracted in the MagNA Pure 96 System in the AMC diagnostic laboratory.
Patient material
pretreatment
viral target
EDTA whole blood
none
CMV, HSV, VZV, EBV
Plasma
none
CSF
none
Respiratory material; resuspend dry swabs in 1 ml
swabs and lavages transport medium
sample volume** elution volume
isolation ­p rotocol
50 µl
50 µl
total NA external lysis
BK, Parvo, HHV8
200 µl
50 µl
total NA external lysis
EV, HPeV, HSV, JC, VZV
200 µl
50 µl
total NA external lysis
EV, HPeV, HRV, InfA, InfB, HAV,
RSV, hMPV, PIV 1-4, hCoV, HBoV
200 µl
50 µl
total NA external lysis
Faeces
pre-lysis of 50 µl faeces in 500 µl EV, HPeV
MP lysisbuffer; Spin down and
use supernatant as input
50 µl
50 µl
total NA external lysis
Biopsy
O/N pre-lysis in 200 µl 1% SDS/ HAV, CMV
prot K solution x 56°C; spin down
and use 100 µl as input
100 µl
50 µl
total NA external lysis
Culture supernatant none
any culturable virus
20 µl
50 µl
total NA external lysis
Vesicle fluid
none
HSV, VZV
20 µl
50 µl
total NA external lysis
Urine
none
BK, CMV
200 µl
50 µl
total NA external lysis
Cytomegalo Virus (CMV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Epstein Bar Virus (EBV), Human Herpes Virus 8 (HHV8), Entero
Virus (EV), Human parEcho Virus (HPeV), Human Rhino Virus (HRV), Influenza A/B (InfA/B), Human Adeno Virus (HAV), Respiratory Syncytial Virus
(RSV), Human Metapneumo Virus (hMPV), Para Influenza Virus (PIV), Human Corona Virus (hCoV), Human Boca Virus (HBoV)
** Final input volume of lysed sample in the MagNA Pure 96 System is 500 µl for all materials
2
3 Materials and Methods
Cerebrospinal fluid (CSF), EDTA whole blood, plasma,
faeces, throat swabs and adenoid tissue biopsies were taken
from the Academic Medical Center clinical virology sample
archives. All patient sample materials used, were collected
according to general hospital standards. We have tested the
daily routine using the MagNA Pure 96 System in our lab by
spiking the samples with viral targets prior to extraction.
Sample types analyzed in our laboratory are summarized in
Table 1. We used different input volumes of samples,
according to the sample type in order to make simultaneous
extraction of a variety of sample types possible.
PCR Controls:
Positive controls were purified plasmids containing the
amplicon sequence of interest. In addition, two different
Internal Controls (IC) were used. Phocine Herpes virus
(PhoHV plasmid) for DNA targets1 and Equine Arteritis
virus (EAV) for RNA targets. 2
Sample Pretreatment
Cerebrospinal Fluid (CSF), EDTA anticoagulated whole
blood and plasma were used without additional
pretreatment.
Throat swabs were resuspended in 2 ml of virus
transportation medium (UTM Universal Transport
Medium, Copan).
Faeces was pre-treated as follows: 50 µl faeces was added
to 450 µl MagNA Pure 96 lysis buffer, mixed, and
subsequently incubated for 10 minutes at room
temperature (+15 to +25°C). The suspension was
centrifuged at 13,000 x g and the supernatant was spiked
with target virus and IC prior to extraction.
Tissue biopsies (ca. 20-40 mm3 in size) were pretreated as
follows: Freshly isolated adenoid tissue was submerged in
1 ml of RNAlater (Ambion) and stored at -15 to -25°C.
Prior to use for extraction RNAlater was pipetted from
the (solid) tissue which was subsequently washed with
PBS; 200 µl of a 1 mg/ml Proteinase K/1% SDS solution
was added to the tissue, and the mixture was incubated
overnight at 56°C. The tube was centrifuged at 13,000 x g
and 100 µl of the supernatant was spiked with Internal
Control (IC), added to 400 µl MagNA Pure 96 lysis buffer
and used for extraction.
input volume of lysed sample was set to 500 µl and
elution was set to 50 µl. Unless otherwise stated, the
guidelines from the package insert were followed.
Purified DNA/RNA using the MagNA Pure 96 System
was used directly in a multiplex PCR using the
LightCycler ® 480 Instrument or, alternatively, in case of
viral RNA targets, subjected to reverse transcription
(RT)-reaction prior to PCR, as described previously.3,4
In brief, 40 µl of the MagNA Pure 96 System eluate (80%
of the original input) was mixed with 10 µl RT-reaction
mix, consisting of 1,500 ng of hexamers, 1x CMB1
buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 0.1%
Triton X-100), 0.4 U of reverse transcriptase per µl, 120
µM (each) deoxynucleoside triphosphate, 0.08 U of
RNAsin per µl, and 5 mM MgCl2. The mixture was
incubated for 30 min at 42°C.
Real-Time PCR Amplification and ­Detection
(using TaqMan ® Probes)
As input in PCR, either 5 µl of the MagNA Pure 96
System eluate (10% of the original input) for DNA or
5 µl of the RT-reaction (8% of the original input) for
RNA was used in a final volume of 20 µl.
In all PCRs performed, the Roche LightCycler ® 480
Probes Master (containing a hot start Taq enzyme) was
used supplemented with 1U/ml Uracil-DNA Glycosylase
(UNG), 900 nM of each primer and 200 nM of each
TaqMan® probe. Sequences of the specific primers and
probes used are published elsewhere. 5,6,7
Detection was done using the Roche LightCycler ® 480
Real-Time PCR Instrument. For all targets the same
PCR program was used with the following cycling
conditions: 2 min x 50°C (activation UNG), 10 min x 95°C
(denaturation template and activation polymerase),
followed by 45 rounds consisting of 15 sec x 95°C
and 1 min x 60°C.
For data analysis with the LightCycler ® 480 Software,
the Second Derivative Maximum analysis method was
used.
DNA/RNA isolation using MagNA Pure 96 System
One single extraction protocol was used for all the different
sample types. All extractions were performed using the
MagNA Pure 96 DNA and Viral NA Small Volume Kit
(Roche Diagnostics), in combination with the Viral NA
Plasma SV external lysis protocol. For all materials, the
3
4 Results
Fluorescence [483-533]
Plasma
21,890 19,890 17,890 15,890 13,890 11,890 9,890 7,890 5,890 3,890 1,890 -0,110 200 µl plasma was spiked with CMV (dilution series 1 x 104
to 1 x 10 2 copies and negative control) and PhoHV IC
(2.5 x 10 3 copies). After extraction the DNA was directly
analyzed in real-time PCR.
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Cycles
Figure 1A shows the amplification curves for CMV (FAM), PhoHV IC
(HEX) in inset.
Fluorescence [483-533]
EDTA whole blood
19,202 17,702 16,202 14,702 13,202 11,702 10,202 8,702 7,202 5,702 4,202 2,702 1,202 -0,298 50 µl EDTA whole blood was spiked with CMV dilution
series (1x 10 4 to 1 x 10 2 copies and negative control) and
PhoHV IC (2.5 x 103 copies). After extraction the DNA
was directly analyzed in real-time PCR.
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Cycles
Figure 1B shows the amplification curves for CMV(FAM), PhoHV IC
(HEX) in inset.
Fluorescence [483-533]
CSF
32,677 29,677 26,677 23,677 20,677 17,677 14,677 11,677 8,677 5,677 2,677 -0,323 200 µl CSF was spiked with HPeV dilution series (1 x 105
to 1 x 101 copies and negative control) and EAV-IC
(2 x ­103 copies). After extraction the RNA was subjected to
RT-reaction and real-time PCR.
Fluorescence [483-533]
2
4
6
Figure 1C shows the amplification curves for HPeV (FAM), EAV IC
(HEX) in inset.
Throat swabs
2,635 2,435 2,235 2,035 1,835 1,635 1,435 1,235 1,035 0,835 0,635 0,435 0,235 0,035 A 200 µl throat swab sample was spiked with Human
Adeno Virus dilution series (1 x 108, 1 x 107, 1 x 105, 1 x 103
and 1 x 101 copies and negative control), HPeV fixed concentration (1 x 103 copies) and PhoHV IC (2.5 x 10 copies).
After extraction the nucleic acid was subjected to RT-reaction
and real-time PCR.
5
4
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Cycles
10
15
20
25
Cycles
30
35
40
45
Figure 1D shows the amplification curves for HAV (LC670), HPeV (FAM;
upper inset) and PhoHV IC (HEX; lower inset) are also shown.
4 Results
Fluorescence [483-533]
Faeces
Faeces (50 µl in bouillon) was pre-lysed in 500 µl MagNA
Pure lysis buffer for 10 min at room temperature
(+15 to +25°C). Samples were spun down for 2 minutes.
Supernatants were spiked with a HPeV dilution series
(1 x 105 to 1 x 101 copies and negative control) and EAV IC
(2 x 103 copies). After extraction, the RNA was subjected
to RT-reaction and real-time PCR.
30,232 27,732 25,232 22,732 20,232 17,732 15,232 12,732 10,232 7,732 5,232 2,732 0,232 2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Cycles
Figure 1E shows the amplification curves for HPeV (FAM), EAV IC
(HEX) in inset.
Fluorescence [483-533]
Biopsies
6 adenoid biopsies, 4 positive and 2 negative for HAV, stored
at -15 to -25°C in RNAlater, underwent pretreatment, as
described above; 100 µl of the biopsy material was spiked
with PhoHV IC (2.5 x 103 copies). For extraction, 100 µl
of the samples was used in the MagNA Pure 96 System.
After extraction, the DNA was directly analyzed using
real-time PCR.
17,023 15,523 14,023 12,523 11,023 9,523 8,023 6,523 5,023 3,523 2,023 0,523 5
10
15
20
25
30
35
40
45
Cycles
Figure 1F shows the amplification curves for HAV (LC670), PhoHV IC
(HEX) in inset.
5 Discussion
We show in this paper that the MagNA Pure 96 System is
able to extract nucleic acid, RNA and/or DNA, from
different kinds of clinical materials. Furthermore, a variety
of sample types could be extracted efficiently in the same
run using the same extraction protocol. To perform these
experiments, we selected patient samples that had been
tested as negative in our diagnostic department. These
samples were spiked with various loads of different viruses,
and subsequently extracted (external lysis protocol) and
used for detection. To all samples, an internal control was
added to exclude inhibition effects and monitor extraction
and PCR efficiencies.
Detection using the Roche LightCycler ® 480 Real-Time PCR
Instrument revealed that extraction from all six different
types of sample materials tested was very efficient. Even
with known difficult materials, such as faeces and biopsies,
the MagNA Pure 96 System performed well.
In addition, we extracted nucleic acid from 2 different
volume inputs (50 and 200 µl) of EDTA whole blood from
patients positive for CMV. Higher inputs led to higher
sensitivity in 7/8 samples, crossing point (Cp) cycle values
were approximately 1.7 cycle lower while Cp values for
IC remained similar (data not shown).
5
6 Conclusion
The MagNA Pure 96 System, in combination with the
MagNA Pure 96 DNA and Viral NA Small Volume Kit, is a
highly efficient and fast system for the extraction of nucleic
acid from a variety of clinical sample materials. Remarkably,
a single extraction protocol combined with a single
downstream real-time RT-PCR protocol can be used. The
MagNA Pure 96 System allows for high-throughput and fast
turnaround times (it takes less than 60 minutes to extract
nucleic acid from 96 samples). Due to the use of a single
generic extraction protocol different materials can be
extracted together simultaneously, thereby adding to the
flexibility of the system. This is especially useful for
molecular diagnostic laboratories that handle large and
heterogeneous sample workflows.
7 References
1
2
3
4
5
6
7
V
an Doornum et al. Journal of Clinical Microbiology, Februari 2003, Vol. 41 no.2; 576
S
cheltinga et al., Journal of Clinical Virology, August 2005, Vol. 33 no. 4; 306
B
eld et al. Journal of Clinical Microbiology, July 2004, Vol. 42 no. 7; 3059
M
olenkamp et al. Journal of Virological Methods, May 2007, Vol. 141 no. 2; 205
B
enschop et al, Journal of Clinical Virology, Februari 2008, Vol. 41 no.2; 69
D
amen et al. Journal of Clinical Microbiology. December 2008. Vol. 46 no.12; 3997.
J ansen et al. Journal of Clinical Virology, May 2011, Epub ahead of print
Important Note: Data included in this article are the sole responsibility of the authors that have published them, and these authors are
responsible for following the respective local regulations for assay setup
and validation.
Potential users are informed to be aware of and in accordance with local
regulations for assay validation and the scope of use for the involved
Roche products, and to ensure that their use is valid in the countries
where the experiments are performed.
Regarding the specific assay(s) described, Roche was neither involved
in establishing the experimental conditions nor in defining the criteria
for the performance of the specific assays. Roche therefore cannot take
any responsibility for performance or interpretation of results obtained
for the biological target parameter(s) described by the authors or other
users using a similar experimental approach.
The MagNA Pure 96 (Cat. Nr. 05195322001) and LightCycler ® 480
Real-Time PCR System are not intended for in vitro diagnostic use
in the U.S.
For life science research only.
Not for use in diagnostic procedures.
MAGNA PURE, LIGHTCYCLER and TAQMAN are trademarks of
Roche. All other product names and trademarks are the property of
their respective owners.
Published by
Roche Diagnostics GmbH
Sandhofer Straße 116
68305 Mannheim
Germany
© 2011 Roche Diagnostics.
All rights reserved.
0611