Biomedical Engineering Reference
In-Depth Information
In this chapter, the two systems discussed as follows are based on the devel-
opment of the quantifications of SARS-CoV RNA in serum. The SARSN RT-
PCR system was designed to amplify the nucleocapsid region and SARSpol1
system was designed to amplify the polymerase region of the virus. These
SARS-CoV RT-PCR systems are useful for the early diagnosis of SARS and
can provide viral load information, helping clinicians to make a prognostic
evaluation of the infected individual.
2. Materials
2.1. Sample Collection
1.
Plain collection tubes for serum collection.
2.
RNase Away (Invitrogen, Carlsbad, CA).
2.2. RNA Extraction
1.
QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany).
2.
Absolute ethanol.
2.3. Real-Time Quantitative RT-PCR
2.3.1. Amplification Reagents
1.
Primers (
see
Note 1
):
a. SARSpol1:
i. Forward: 5'-GAGTGTGCGCAAGTATTAAGTGA-3'.
ii. Reverse: 5'-TGATGTTCCACCTGGTTTAACA-3'.
b. SARSN:
i. Forward: 5'-TGCCCTCGCGCTATTG-3'.
ii. Reverse: 5'-GGCCTTTACCAGAAACTTTGC-3'.
2.
Dual-labeled fluorescent probes (
see
Note 1
):
a. SARSpol1:
5'-(FAM)ATGGTCATGTGTGGCGGCTCACTA(TAMRA)-3'.
b. SARSN:
5'-(FAM)TGCTAGACAGATTGAACCAGCTTG(TAMRA)-3',
where FAM is 6-carboxy-fluorescein; TAMRA is 6-carboxy-tetramethyl-
rhodamine.
The amplicon locations of the SARSN and SARSpol1 RT-PCR systems are
shown in
Fig. 1
.
3.
Calibrators:
a. Synthetic DNA oligonucleotide corresponding to part of the polymerase gene
of SARS-CoV genome, HPLC-purified (Proligos, Singapore) (
see
Note 2
):
5'-AACGAGTGTGCGCAAGTATTAAGTGAGATGGTCATGTGTGG
CGGCTCACTATATGTTAAACCAGGTGGAACATCATCCGG-3'.
