Quantification of SARS Coronavirus RNA by Real-Time PCR for Rapid Detection of Anti-viral Activity
Date Issued
2004
Date
2004
Author(s)
Wang, Yu-Zhu
DOI
zh-TW
Abstract
Severe Acute Respiratory Syndrome (SARS) has threatened the health of people around the world because of its high transmission and high mortality rate. It is caused by a novel coronavirus, named as SARS coronavirus (SARS-CoV). There are no efficient and specific strategies to treat and prevent SARS up to now. Therefore, it is very urgent and important to develop vaccines and drugs against SARS. Neutralization test is the standard method to evaluate whether or not the sera or drugs have neutralizing activity. However, conventional method is too time-consuming. In this study, a rapid method using real-time polymerase chain reaction (PCR) was established to detect anti-SARS-CoV activity.
Real-time PCR was utilized to detect the RNA of SARS-CoV. Probes were designed for the target genes, RNA-dependent RNA polymerase (RdRP) and nucleocapsid (NC) genes. Vero E6 cells were inoculated with SARS-CoV and subsequently the amounts of SARS-CoV RNA were detected at different time points. The amounts of viral RNA could be detected at significant level after 8 hours. Moreover, the amounts detected for plus-sense RNA and minus-sense RNA were similar. Our data also showed that the detection for the NC RNA was not more sensitive than that for the RdRP RNA. Furthermore, the neutralization tests were performed for the sera from patients in the convalescent phase or from mice immunized with synthetic peptides of SARS-CoV. The results showed that no viral RNA could be detected for the SARS-CoV pretreated with sera with neutralizing activity. This study revealed that using real-time PCR could detect the neutralizing activity against SARS-CoV more rapidly.
RNA interference (RNAi) is thought to have the potential to fight against virus infection. This study also attempted to develop small interfering RNA (siRNA) with the ability to inhibit the growth of SARS-CoV. First, siRNA targeting the NC gene of SARS-CoV was produced by in-vitro transcription and using DNA vector. After SARS-CoV was inoculated into Vero E6 cells which had been transfected by siRNA, the cytopathic effect was observed. Unfortunately, the preliminary results were not as expected. In the future, the condition of experiments should be optimized to improve the efficiency of inhibition and more target genes for siRNA could be designed to inhibit the growth of SARS-CoV.
Subjects
連鎖反應
急性嚴重呼吸道症候群
即時聚合酶
中和試驗
SARS
Neutralization test
real-time polymerase chain reaction
SDGs
Type
other
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