Biomedical Engineering Reference
In-Depth Information
b.
Synthetic DNA oligonucleotide corresponding to part of the nucleocapsid gene
of SARS-CoV genome, HPLC-purified (Proligos, Singapore) (
see
Note 2
):
5'-GAAACTGCCCTCGCGCTATTGCTGCTAGACAGATTGAACC
AGCTTGAGAGCAAAGTTTCTGGTAAAGGCCAACAA-3'.
4.
RNase-free water.
5.
EZ r
Tth
RNA PCR reagent set (Applied Biosystems, Foster City, CA).
2.3.2. Instrumentation for Quantitative Analysis
ABI Prism 7700 Sequence Detector (Applied Biosystems).
3. Methods
3.1. Prevention of Contamination
Because of the high sensitivity of RT-PCR-based approaches, strict precau-
tions should be applied to prevent the RT-PCR assay from contamination
(
22
)
.
These precautions include:
1.
Aerosol-resistant pipet tips should be used for all liquid handling.
2.
Separate areas should be used for the RNA extraction step, the setting up of
amplification reactions, the addition of template, and the carrying out of amplifi-
cation reactions.
3.
Real-time PCR approaches obviate the need for post-PCR processing and further
reduce the risk of contamination.
4.
The assay should include a further level of anticontamination measure in the
form of pre-amplification treatment using uracil
N
-glycosylase, which destroys
any carried-over uracil containing PCR products
(
23
)
.
5.
Multiple negative water blanks should be included in every analysis so as to detect
the possibility of reagent contamination.
3.2. Sample Collection
1.
Collect blood samples in plain tubes. To ensure a sufficient amount of serum for
RNA analysis, at least 3 mL of blood should be taken for each sample (
see
Note 3
).
The blood samples should be processed as soon as possible to maximize the
chance of obtaining good-quality viral RNA. If the processing procedures cannot
take place immediately, the blood samples should be stored with extra care
(
see
Note 4
).
2.
Centrifuge the blood samples at 1600
g
for 10 min at 4°C.
3.
Transfer serum into new tubes.
4.
Store the serum at -80°C until RNA extraction (
see
Note 5
).
3.3. RNA Extraction
The RNA extraction should be performed in a clean and separate area to
minimize the chance of cross-contamination.
