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EPICENTRE Forum 5 (3)
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Judith T. Schanke, John Watson, and Haiying L. Grunenwald, Epicentre
Technologies
The use of PCR for the detection of blood-borne microbes has increased
significantly in recent years. Hepatitis C virus (HCV), present in human
whole blood and serum samples, is now identified quickly and easily by
isolating nucleic acids and subsequently amplifying a conserved region
of the RNA virus by RT-PCR. For low copy number samples, a subsequent
nested PCR amplification can generate sufficient product for accurate
detection. Similarly, bacteria present in a human plasma sample are now
detected by DNA isolation and subsequent amplification of a definitive
bacterial gene.
Here, we demonstrate the efficiency and ease of isolating nucleic acids
from blood-derived samples using the new MasterPure™ Complete DNA
and RNA Purification Kit.* Total nucleic acid, including
both DNA and RNA, was isolated in less than one hour from HCV-containing
whole blood and serum samples. The conserved 5' noncoding region of HCV,
which is commonly used to classify HCV into genotypes and subtypes, was
subsequently amplified using the MasterAmp™ RT-PCR Kit. Total nucleic
acid was also efficiently isolated from human plasma that contained E.
coli, as was demonstrated by the amplification of a region of the E.
coli 16S rRNA gene.
HCV serum samples
Human serum samples containing HCV were obtained from Dr. Yury Khudyakov
and Dr. Howard Fields, Hepatitis Branch, Centers for Disease Control,
Atlanta, GA. Each sample was provided with a viral titer. These titers
were used to calculate the maximum possible copy number of virus used
in amplification reactions assuming 100% yield of viral RNA.
Introduction of HCV into whole blood
In order to simulate the isolation of HCV RNA from whole blood, two
hundred microliters of non-infected whole blood were centrifuged and
75 µl of plasma were removed. The plasma was replaced with 75 µl
of serum containing HCV RNA. This combined blood sample was then vortex
mixed to resuspend the pelleted cells and the MasterPure Complete DNA
and RNA Purification Kit was used to isolate nucleic acids from this
whole blood sample.
Rapid isolation of DNA and RNA from HCV-containing whole blood
A total nucleic acid extraction was performed on a small aliquot (12.5 µl)
of the HCV-containing whole blood sample using Epicentre's MasterPure
Complete DNA and RNA Purification Kit according to the protocol described
in Table 1. This volume of whole blood was used to facilitate nucleic
acid isolation in a single 1.5-ml tube. This procedure is rapid and does
not entail lysis and removal of the red blood cells before nucleic acid
isolation. The precipitated nucleic acid obtained from this procedure,
which contains both genomic DNA as well as total cellular and viral RNA,
was resuspended in 25 µl of TE buffer. Five microliters of the
sample were separated by agarose gel electrophoresis, stained with ethidium
bromide, and visualized by transillumination (Figure 1A). The sample
contains a prominent genomic DNA band. Ribosomal RNA bands are not apparent;
however, the sample clearly contains viral RNA as demonstrated by the
RT-PCR analysis described below.
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Table 1. MasterPure Complete Kit Protocol for
Extraction of Nucleic Acids from Whole Blood.
- Add 12.5 µl of whole blood to 400 µl of Tissue
and Cell Lysis Buffer containing 2 µl of 50 µg/µl
Proteinase K per sample.
- Incubate 15 minutes at 65°C. Vortex mix every 5 minutes.
- Place on ice for 5 minutes.
- Add 225 µl of Protein Precipitation Reagent and vortex
mix for 10 seconds.
- Spin 10 minutes in a microcentrifuge.
- Transfer the supernatant, containing both DNA and RNA, to another
tube.
- Add 600 µl of isopropanol and invert the tube to mix,
approximately 30-40 times.
- Spin 10 minutes in a microcentrifuge at 4°C. Decant the
isopropanol.
- Wash the pellet twice with 70% ethanol and remove the last
traces of ethanol.
- Resuspend the pellet in 10-50 µl of TE buffer.
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Figure 1. Isolation and detection of HCV RNA from whole blood.
Panel A, Electrophoresis of DNA and RNA isolation from HCV-containing
whole blood. Lane 1, 1 kb ladder; Lane 2, total nucleic acid isolated
from whole blood. Panel B, RT-PCR of HCV RNA isolated from
whole blood. Lane 1, 100 bp ladder; Lane 2, 296 bp HCV RT-PCR product. Panel
C, Nested PCR amplification of 232 bp HCV RT-PCR product. Lane
1, 100 bp ladder; Lane 2, nested amplification product of a 1:10
dilution of the HCV RT-PCR product; Lane 3, nested amplification
product of a 1:100 dilution of the HCV RT-PCR product. |
Amplification of HCV RNA from whole blood nucleic acid isolation
To verify that the HCV RNA had been isolated along with human total
nucleic acid, the MasterAmp RT-PCR Kit was used to amplify a 296 bp region
of the highly conserved 5' noncoding region of HCV. The RT-PCR reaction
contained 5 µl (containing 300-400 copies of HCV) of the isolated
total nucleic acid sample, 1X RT-PCR Buffer, 3 mM MgCl2, 0.5
mM MnSO4, 2X MasterAmp PCR Enhancer (with betaine+),
12.5 pmoles each of HCVRT-F and HCVRT-R primers (Table 2), 400 µM
each dNTP, and 2.5 units of RetroAmp™ RT DNA Polymerase. Reverse
transcription was performed at 60°C for 20 minutes, followed by 40
cycles of 92°C for 30 seconds, 55°C for 1 minute, and 72°C
for 1 minute. Ten microliters of the RT-PCR product were separated on
a 2% agarose gel, stained with SYBR® Gold (Molecular Probes), and
visualized by transillumination (Figure 1B). The 296 bp product amplified
from approximately 300 copies of HCV was easily detectable.
Table 2. Primer Sequences.
HCVRT-F 5'-CTGTGAGGAACTACTGTCTTC-3'
HCVRT-R 5'-GGTGCACGGTCTACGAGACCT-3'
HCVIN-F 5'-CAGAAAGCGTCTAGCCATGGCGTT-3'
HCVIN-R 5'-CCCTATCAGGCAGTACCACAA-3'
16S rRNA-F 5'-CTGCTGCCTCCCGTAGGAGT-3'
16S rRNA-R 5'-AGAGTTTGATCCTGGCTCAG-3' |
Abundant product yield obtained from nested amplification of the
HCV 5' noncoding region
For further amplification and to increase confidence in the detection
of the HCV product, a second, nested PCR was performed using primers
internal to the HCVRT primers used above for the RT-PCR. The RT-PCR product
was diluted 1:10 or 1:100 in water and 1 µl of each dilution was
used in a nested PCR. Each reaction contained 1X PCR buffer, 2.5 mM MgCl2,
2X MasterAmp PCR Enhancer (with betaine), 200 µM each dNTP, 12.5
pmoles each of HCVIN-F and HCVIN-R primers (Table 2), 1 µl of each
diluted RT-PCR product, and 1.25 units of MasterAmp Taq DNA Polymerase
in a total volume of 25 µl. These nested reactions were amplified
with 20 cycles of the following profile: 94°C for 30 seconds, 64°C
for 1 minute, and 73°C for 1 minute. Two microliters (8%) of each
product were separated by agarose gel electrophoresis. The gel was stained
with SYBR Gold and visualized by transillumination. The prominent HCV
nested amplification products were obtained even with a 100-fold template
dilution (Figure 1C). In some cases of low viral titers, it may be necessary
to perform such a nested PCR to detect the presence of the virus.
Abundant amplification of HCV RNA purified from clinical serum samples
Typically, viruses such as HCV are isolated from human serum instead
of whole blood, as was performed above. The MasterPure Complete DNA and
RNA Purification Kit was used to isolate total nucleic acid from 50 µl
of each of two HCV-containing serum samples. The resulting nucleic acid
pellet, containing cellular DNA and total cellular and viral RNA, was
resuspended in 50 µl of TE buffer. Five microliters of each sample
(containing approximately 800-900 copies of HCV) were then subjected
to RT-PCR using the conditions described above, but using only 30 cycles
for the amplification. Ten percent of each reaction was separated by
agarose gel electrophoresis, stained with SYBR Gold, and visualized by
transillumination. The 296-bp amplification product of the 5' noncoding
region of HCV is readily detectable when isolated from the serum (Figure
2).
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Figure 2. Amplification of HCV RNA isolated from human serum. Lane
1, 100 bp ladder; Lane 2, HCV serum sample A; Lane 3, HCV serum sample
B. |
Detection of bacterial DNA in human plasma
Cells from a single E. coli colony were used to introduce intact E.
coli into 50 µl of human plasma. This model system was created
to determine if the MasterPure Complete Kit could be used to isolate
both the human and E. coli DNA, and to demonstrate that the E.
coli DNA could be detected in the nucleic acid mixture. Total nucleic
acid was isolated from the plasma sample using the standard protocol
of the MasterPure Complete DNA and RNA Purification Kit.1 The
pellet, containing both DNA and RNA, was resuspended in 50 µl
of TE buffer. Fifteen microliters of the sample were separated by agarose
gel electrophoresis, stained with ethidium bromide, and visualized
by transillumination. The pure, high molecular weight genomic DNA and
the prominent ribosomal RNA isolated can be seen in Figure 3A.
One microliter (2% of the DNA isolated) of the human plasma total nucleic
acid was used to amplify a 350 bp region of the 16S ribosomal gene of E.
coli. One nanogram of purified E. coli DNA was used as a positive
control for PCR amplification. The amplification reactions included 1X
PCR buffer, 3 mM MgCl2, 1X MasterAmp PCR Enhancer (with betaine),
200 µM each dNTP, 50 pmoles of each 16S rRNA primer (Table 2),
1.25 units of MasterAmp Taq DNA Polymerase, and 1 µl of
isolated sample or 1 ng of E. coli DNA control template. A reaction
with no DNA template, a negative control for the detection of E. coli DNA
contamination, was also performed. The samples were heated at 94°C
prior to addition of MasterAmp Taq Polymerase. Then the reactions
were denatured for 4 minutes at 94°C and cycled 25 times with the
following conditions: 94°C for 1 minute, 55°C for 1 minute, and
72°C for 1 minute. Five microliters of each sample were separated
by agarose gel electrophoresis, stained with ethidium bromide, and visualized
by transillumination (Figure 3B). The 350 bp product is clearly detectable
in both the positive control and the E. coli-contaminated human
DNA sample, but not in the no DNA control. The MasterPure Complete DNA
and RNA Purification Kit was used to successfully isolate E. coli genomic
DNA from human plasma.
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Figure 3. Quick isolation and detection of E. coli DNA
from human plasma. Panel A, DNA and RNA isolation of human
blood contaminated with E. coli. Lane 1, kb ladder; Lane
2, total nucleic acid isolated from whole blood. Panel B, Detection
of the 16S ribosomal RNA gene from E. coli in DNA isolated
from E. coli-contaminated human plasma. Lane 1, 100 bp ladder;
Lane 2, no DNA template control amplification; Lane 3, 1 ng of E.
coli DNA template; Lane 4, 1 µl of DNA isolated from
human plasma contaminated with E. coli. |
Nucleic acid from HCV-containing whole blood samples can be purified
with the MasterPure Complete DNA and RNA Purification Kit in less than
one hour, and HCV RNA can be easily detected from the resulting total
nucleic acid sample with Epicentre's MasterAmp RT-PCR Kit. HCV RNA can
be detected similarly from human serum samples. Performing a second nested
PCR on the HCV RT-PCR product using MasterAmp Taq DNA Polymerase
improves the sensitivity of virus detection. In addition, bacterial DNA
can easily be amplified from nucleic acid isolated from E. coli-contaminated
plasma samples isolated with the MasterPure Complete DNA and RNA Purification
Kit; therefore blood-borne microbes would likely be detectable from these
samples using PCR.
- Schanke, J.T. and Watson, J. (1998) Epicentre Forum 5 (2),
12.
MasterPure™ Complete DNA and RNA
Purification Kit
MasterAmp™ High Fidelity RT-PCR Kit
MasterAmp™ RT-PCR Kit for High Sensitivity
*Patents pending.
+Patents issued and pending.
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