Biomedical Engineering Reference
In-Depth Information
16
Genomic Sequencing of the Severe Acute Respiratory
Syndrome-Coronavirus
Stephen S. C. Chim, Rossa W. K. Chiu, and Y. M. Dennis Lo
Summary
The polymerase chain reaction (PCR), which can exponentially replicate a target
DNA sequence, has formed the basis for the sensitive and direct examination of clinical
samples for evidence of infection. During the epidemic of severe acute respiratory syn-
drome (SARS) in 2003, PCR not only offered a rapid way to diagnose SARS-coronavirus
(SARS-CoV) infection, but also made the molecular analysis of its genomic sequence
possible. Sequence variations were observed in the SAR-CoV obtained from different
patients in this epidemic. These unique viral genetic signatures can be applied as a pow-
erful molecular tool in tracing the route of transmission and in studying the genome
evolution of SARS-CoV. To extract this wealth of information from the limited primary
clinical specimens of SARS patients, we were presented with the challenge of efficiently
amplifying fragments of the SARS-CoV genome for analysis. In this chapter, we will
discuss how we managed to accomplish this task with our optimized protocols on reverse-
transcription, nested PCR amplification, and DNA cycle sequencing. We will also dis-
cuss the sequence variations that typified some strains of SARS-CoV in the different
phases during this epidemic. PCR amplification of the viral sequence and genomic
sequencing of these critical sequence variations of re-emerging SARS-CoV strains would
give us quick insights into the virus.
Key Words:
SARS coronavirus; viral RNA extraction; reverse-transcription PCR;
sequencing; genomic sequence variation.
1. Introduction
Severe acute respiratory syndrome-coronavirus (SARS-CoV), the etiologic
agent of SARS
(
1-3
)
, is a virus that was unknown to us before the SARS
epidemic. The concerted efforts of researchers have promptly elucidated its
genetic code. The genome of SARS-CoV is a 29,727-nucleotide, polyadenyl-
ated RNA. The genomic organization is typical of coronaviruses, having the
characteristic gene order (5'-
polymerase
[
Orf1ab
],
spike
[
S
],
envelope
[
E
],
mem-
