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Figure 1. Replication-specific multiplex reverse transcriptase–polymerase
chain reaction (RT-PCR) assay shows severe acute respiratory syndrome–associated
coronavirus (SARS-CoV) replicated in the lungs and intestines of mice.
A) Vero E6, murine fibroblast (L2), and human lung fibroblasts (MRC5)
were inoculated with SARS-CoV at an MOI of ≈0.001 or were mock-inoculated
(M). G3PDH, SARS-CoV gRNA, and sgRNA were amplified by multiplex RT-PCR
from total RNA extracted at 1 h, 16 h, or 5 days after inoculation. Amplicons
were visualized by ethidium bromide staining after electrophoresis; negative
images are shown. B) Mice were inoculated with 2x105 50% tissue
culture infective dose of SARS-CoV (lanes labeled 6–17) or were mock-inoculated
(M) and euthanized after 3, 5, 7, or 10 days. G3PDH, SARS-CoV gRNA, and
sgRNA were amplified by multiplex RT-PCR from total RNA extracted from
the lung (L) and intestine (I) harvested at various time points. Heminested
PCR (HN) was used to amplify gRNA and sgRNA from RT-PCR reactions. Positive
and negative controls for PCR reactions are indicated by + and –, respectively.
D indicates DNA marker ladder. *The doublet observed in HN-PCR reactions
results from residual primers used in the primary amplification reaction.
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