Download Performance Efficient Extraction of Viral DNA and RNA from a

MagNA Pure System Application Note No. 1
Performance Efficient Extraction
of Viral DNA and RNA from a
Variety of Human Sample
Materials
using a Single Standardized Protocol
with the MagNA Pure 96 System
René Minnaar and Richard Molenkamp
Department of Medical Microbiology,
March 2015
Abstract
In our 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 sample materials.
Extraction was very sensitive and effi cient, 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.
Academic Medical Center, (AMC),
Amsterdam, The Netherlands
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.
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 medicalmicrobiology laboratories has increased
significantly. Not only has the amount of samples processed and the choice of targets increased, the nature and
variety of the sample materials to be tested have grown.
Testing requires 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 sample 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.
Materials and Methods
Virus stocks and patient material
DNA viruses Human Adenovirus (HAV) and
Cytomegalovirus(CMV), as well as RNA viruses Human
Parechovirus(HPeV) and Influenza A virus, were isolated
and cultured from sample materials sent to the laboratory for
molecular diagnosis. Virus stocks were TCID50* quantified.
*Median tissue culture infective dose; the amount of a cytopathogenic
agent (virus) that will produce a cytopathic effect in 50% of cell cultures
inoculated, expressed as TCID50/ml.
Table 1: Treatment of various human sample materials to be extracted in the MagNA Pure 96 System in the AMC diagnostic laboratory.
Material
Pretreatment
Viral Target
Sample Volume** Elution Volume Isolation Protocol
EDTA whole blood
none
CMV, HSV, VZV, EBV
50 μl
50 μl
total NA external lysis
Plasma
none
BK, Parvo, HHV8
200 μl
50 μl
total NA external lysis
CSF
none
EV, HPeV, HSV, JC, VZV
200 μl
50 μl
total NA external lysis
Respiratory material;
swabs and lavages
resuspend dry swabs in
1 ml transport medium
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 MP lysisbuffer; Spin down and use
supernatant as input
EV, HPeV
50 μl
50 μl
total NA external lysis
Biopsy
O/N pre-lysis in 200 μl
1% SDS/prot K solution
x 56°C; spin downand
use 100 μl as input
HAV, CMV
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
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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 human 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 differentInternal 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.
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 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.
To assess the MagNA Pure 96 System utility, multiplex
PCR using the LightCycler® 480 Instrument was used to
analyze MagNA Pure 96-purified DNA/RNA 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.
3
Results
EDTA whole blood
21,890 -
19,202 -
19,890 -
17,702 -
Fluorescence (483–533)
17,890 15,890 13,890 11,890 9,890 7,890 5,890 3,890 -
16,202 14,702 13,202 11,702 10,202 8,702 7,202 5,702 4,202 2,702 1,202 -
-0,110 -
-0,298-
-
1,890 -
Cycles
Cycles
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Figure 1b. Shows the amplification curves for CMV(FAM), PhoHV IC
(HEX) in inset.
EDTA whole blood. 50 μl EDTA whole blood was spiked with CMV dilution
series (1x 104 to 1 x 102 copies and negative control) and PhoHV IC (2.5 x 103
copies). After extraction the DNA was directly analyzed in real-time PCR.
2,635 -
29,677 -
2,435 -
14,677 11,677 8,677 5,677 -
1,835 1,635 1,435 1,235 1,035 0,835 0,635 0,435 -
2,677 -
0,235 -
-0,323 -
0,035 -
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Cycles
Figure 1c. Shows the amplification curves for HPeV (FAM), EAV IC
(HEX) in inset.
CSF. 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.
5
10
15
20
25
Cycles
30
-
17,677 -
2,035 -
-
20,677 -
2,235 -
-
23,677 -
-
Fluorescence (483–533)
26,677 -
2
4
Throat swabs
32,677 -
-
Fluorescence (483–533)
CSF
-
Figure 1a. Shows the amplification curves for CMV (FAM),
PhoHV IC (HEX) in inset.
Plasma. 200 μl plasma was spiked with CMV (dilution series 1 x 104 to
1 x 102 copies and negative control) and PhoHV IC (2.5 x 103 copies).
After extraction the DNA was directly analyzed in real-time PCR.
2
-
6
-
4
-
2
-
Fluorescence (483–533)
Plasma
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.
Throat swabs. 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.
Results
EDTA whole blood
30,232 -
17,023 -
27,732 -
15,523 -
5,023 3,523 -
2,732 -
2,023 -
0,232 -
0,523 -
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.
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.
5
10
15
20
Cycles
25
30
-
5,232 -
6,523 -
-
7,732 -
-
10,232 -
8,023 -
-
12,732 -
9,523 -
-
15,232 -
11,023 -
-
17,732 -
12,523 -
-
20,232 -
14,023 -
-
22,732 -
-
Fluorescence (483–533)
25,232 -
-
Fluorescence (483–533)
Faeces
35
40
45
Figure 1f. Shows the amplification curves for HAV (LC670), PhoHV IC
(HEX) in inset.
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.
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 samples. 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 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.
Discussion 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
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.
References
1.Van Doornum et al. Journal of Clinical Microbiology,
Februari 2003, Vol. 41 no.2; 576
2. S cheltinga et al., Journal of Clinical Virology, August
2005, Vol. 33 no. 4; 306
3.B eld et al. Journal of Clinical Microbiology, July 2004,
Vol. 42 no. 7; 3059
4.Molenkamp et al. Journal of Virological Methods, May
2007, Vol. 141 no. 2; 205
Important Note: 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.
5.B enschop et al, Journal of Clinical Virology, Februari
2008, Vol. 41 no.2; 69
www.ehec.org/pdf/Laborinfo_01062011.pdf
6.Damen et al. Journal of Clinical Microbiology. December
2008. Vol. 46 no.12; 3997.
7.Jansen et al. Journal of Clinical Virology, May 2011, Epub
ahead of print
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.
The MagNA Pure 96 Instrument (06 541 089 001) is for in vitro
diagnostic use.
The LightCycler ® 480 Instrument is for life science research
only. Not for use in diagnostic procedures.
6
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