Molecular Diagnosis of Severe Acute Respiratory Syndrome - Clinical Applications of PCR - page 165

Fig. 1. The locations of the polymerase chain reaction (PCR) amplicons within the
genomic organization of severe acute respiratory syndrome (SARS)-coronavirus
(CoV). The black bars represent the two PCR amplicons located within the genomic
structure of SARS-CoV. The size of the open reading frames (ORFs) is drawn to scale,
except for orf1ab. The size of the genome is 29.3 kb. Shaded boxes represent ORFs
encoding the viral polymerase, whereas the filled box (N) represents the nucleocapsid
region. S represents spike protein; E represents envelope protein; M represents mem-
brane protein.
these data can be difficult because of the inability to standardize such data as a
result of numerous factors, such as sampling technique for nasopharyngeal
aspirates, urine volume, variations of bowel transit time (e.g., during diarrhea),
or stool consistency. On the other hand, plasma/serum-based assays may allow
the precise and standardized quantitative expression of viral loads, thus
enabling the assessment of disease severity and prognosis. Detection of viral
nucleic acids in plasma/serum has been well established for viral load studies
for numerous other viruses ( 16 , 17 ) . At the beginning of the SARS outbreak, a
single report showed the relatively low sensitivity of detecting SARS-CoV
RNA in plasma using an ultracentrifugation-based approach, with low concen-
trations of SARS-CoV detected in the plasma of a patient 9 d after disease
onset ( 2 ) . Subsequently, together with the improvement of viral RNA extrac-
tion in which plasma or serum requires no ultracentrifugation, two real-time
quantitative RT-PCR assays, one aimed toward the polymerase region and the
other toward the nucleocapsid region of the virus genome ( Fig. 1 ), were devel-
oped for measuring the concentration of SARS-CoV RNA in serum/plasma
samples from SARS patients ( 13 , 14 ) . In these assays, the absolute calibration
curves are constructed by serial dilutions of high-performance liquid chroma-
tography (HPLC)-purified, single-stranded synthetic DNA oligonucleotides
specifying the studied amplicons ( Fig. 2 ). Previous studies have shown that
such single-stranded oligonucleotides reliably mimic the products of the
reverse-transcription step and produce calibration curves that are identical to
those obtained using T7-transcribed RNA ( 18 , 19 ) . The use of such calibration
methodology significantly simplifies the process of obtaining a calibration
curve when compared with the labor-intensive preparation of calibration curve
involving amplicon subcloning and in vitro transcription.