Biomedical Engineering Reference
In-Depth Information
Table 1
Composition of Reverse-Transcription Polymerase Chain Reaction Mix
for Amplification of Pol and N Genes of Severe Acute Respiratory
Syndrome-Coronavirus
Component
Volume for one reaction (µL)
Final concentration
5X TaqMan EZ buffer
5
1X
Mn(OAc)
2
(25 m
M
)
3
3 m
M
dATP (10 m
M
)
0.75
300 µ
M
dCTP (10 m
M
)
0.75
300
µ
M
dGTP (10 m
M
)
0.75
300
µ
M
dUTP (20 m
M
)
0.75
600 µ
M
Forward primer (10
µ
M
)
0.5
300 n
M
Forward primer (10
µ
M
)
0.5
300 n
M
Probe (5 µ
M
)
0.5
100 n
M
r
Tth
DNA Polymerase (2.5 U/
µ
L)
1
0.1 U/
µ
L
AmpErase uracil-
N
-glycosylase
(1 U/µL)
0.25
0.01 U/µL
Total volume
13.75
Table 2
Cycling Profile for Amplification of Pol and N Genes
of Severe Acute Respiratory Syndrome-Coronavirus
Step
Temperature
Time
Uracil
N
-glycosylase (UNG) treatment
50°C
2 min
Reverse transcription
60°C
30 min
Deactivation of UNG
95°C
5 min
Denaturation
94°C
20 s
40 Cycles
Annealing/extension
56°C
1 min
extraction (typically 50
µ
L),
V
Serum
represents the volume of serum extracted
(typically 0.28 mL).
The validations of the two real-time RT-PCR systems are as described previ-
ously
(
13
)
.
4. Notes
1.
Primers and probes are designed with the use of the
Primer Express
®
Software
v2.0 (Applied Biosystems). Certain precautions for the design are listed as
follows:
