Question

4a. Using SARS-CoV as a specific example, describe how epidemiologists can track infections during an
epidemic by a) measuring antibody levels (seroconversion) and b) using PCR methods.
4b. During the course of a single infection, which method will detect SARS-CoV earliest? Explain why.

Answer both questions thoroughly and completely

Answer 1

4a During the SARS-CoV outbreak, rapid diagnosis was best made by RT-PCR using defined primers, usually derived from the viral N sequence. For early diagnosis, specimens from throat or nasal pharyngeal swabs can be used for RT-PCR, and serum samples were also used to detect viral RNA during the first week of illness. Both stool and respiratory specimens were assayed for viral RNA during the second week of illness. In contrast, serological assays usually provide the best way to confirm or rule out infections ex post facto. EIAs for viral N and S antigens were also developed to screen for suspected patients in rural areas.

RT-pCR RNA extraction is performed by using QIAamp Viral RNA kit reagents. The RT-PCR primers and conditions have been described in kits. Since these primers gave occasional false-positive reactions with stool specimens, all PCR-positive stool specimens were retested by the LightCycler PCR for confirmation using the same two sets of primers, with the melting curve analysis being used to provide additional confirmation of reaction specificity. A plasmid vector pCRII-TOPO containing the RNA-dependent RNA polymerase-encoding sequence of the virus was used as the reference standard. A series of five log10 dilutions corresponding to 1 x 101 to 1 x 106 copies per reaction of reference standard was run in parallel with the test samples.

Antibody testing: Coronavirus immunoglobulin G serologic testing is performed by indirect immunofluorescence. Batches of SARS-CoV–infected Vero cell smears were prepared and fixed in ice-cold acetone for 10 minutes. The cells were adjusted to be 60% to 70% SARS-CoV infected, as judged by immunofluorescent staining with a control positive human convalescent-phase serum. The fixed smears were stored at –70°C until use. Serum samples were screened at a dilution of 1:10 on infected and uninfected control cells. After 30 minutes of incubation, the cells were washed twice in phosphate-buffered saline (PBS) for 5 minutes each, and then goat anti-human fluorescein isothiocyanate conjugate was added, and the cells were incubated for 30 minutes at 37°C. The cells were washed again as described and examined with an immunofluorescent microscope. Serum samples positive at a screening dilution of 1:10 were titrated with serial twofold dilutions in parallel with the respective acute-phase serum specimen from the same patient. A positive control serum was tested with each batch of cells.

4b RT PCR is used to detect SARC COV by taking swap and doing PCR for Viral N sequence.By optimizing RNA extraction methods and applying quantitative real time RT-PCR technologies, the sensitivity of tests for early diagnosis of SARS can be greatly enhanced.Whereas antibody takes 7 to 11 days for better diagnosis.