Biomedical Engineering Reference
In-Depth Information
1.
Add 1.12 mL of AVL buffer to 0.28 mL of serum, mix, and incubate at room
temperature for 10 min.
2.
Add 1.12 mL of absolute ethanol to the mixture and mix.
3.
Apply the mixture to an RNeasy mini column and wash the column according to
the manufacturer's recommendations.
4.
To elute RNA, add 50
L of AVE buffer onto the silica-gel membrane and
incubate for 1 min at room temperature. Centrifuge the RNeasy column for 1 min
at 6000
g
.
µ
5.
Store the extracted RNA at -80°C until use.
3.4. Real-Time Quantitative RT-PCR
Serum SARS-CoV RNA is quantified by using one-step real-time quantita-
tive RT-PCR
(
24
)
. In this method, the r
Tth
(
Thermus thermophilus
) DNA poly-
merase functions both as a reverse transcriptase and a DNA polymerase
(
25
)
(
see
Note 7
). Instead of using a two-step RT-PCR approach, one-step
RT-PCR, incorporating both the reverse transcription and PCR steps in a single
tube, should be used to reduce both hands-on time and the risk of contamina-
tion. In this protocol, the quantification of SARS-CoV RNA is described as
follows.
3.4.1. Quantification of pol and N Genes of SARS-CoV Genome
1.
Prepare calibration curves by serially diluting the synthetic DNA oligonucleotides
specifying the amplicons with concentrations ranging from 1.0
×
10
7
copies to 5
copies (
see
Note 3
).
2.
Set up the RT-PCR reaction mixture for the
Pol
and
N
genes according to
Table 1
.
3.
Add 11.25
L of sample RNA, synthetic DNA oligonucleotides (for calibration
curve), or RNase-free water (for negative blanks) into the reaction mixture.
µ
4.
Perform the real-time RT-PCR reactions in the ABI Prism 7700 Sequence Detec-
tor with cycling conditions shown in
Table 2
.
3.4.2. Data Analysis
Amplification data are analyzed and stored by the Sequence Detection Sys-
tem Software (v1.9; Applied Biosystems). The SARS-CoV RNA concentra-
tion is expressed as copies per milliliter of serum (copies/mL). The calculation
is shown as follows:
CQ
V
V
RNA
=×
Plasma
in which
C
represents the SARS-CoV RNA concentration in serum (copies/
mL),
Q
represents the target quantity (copies/
L) determined by a sequence
detector in a PCR,
V
RNA
represents the total volume of RNA obtained after
µ
