Novel Coronavirus Laboratory Diagnostic Methodology

The initial case of Severe Acute Respiratory Syndrome (SARS) was found in 11 November 2002 in Guangdong Province, South China. The disease spread throughout China and to other areas of the world rapidly. According to WHO, there was a cumulative number of 8450 cases with 727 deaths in 33 countries and regions globally during the SARS epidemic. After a more though and comprehensive research carried out by the multi-laboratory international network, WHO officially announced that this new coronavirus, provisionally termed SARS-associated coronavirus (SARS CoV), was the causal agent of SARS.SARS CoV is a newly identified member of the Coronoviridae family of enveloped, positive-stranded RNA viruses. Four putative structural genes, i.e. spike protein(S), envelop protein (E), membrane protein (M) and nucleocapsid protein (N) could be identified. Research in the past year has revealed that S, N, and M protein can induce immunogenicity. The N protein as an immunogen in particular is now believed to be the best choice for antibody-oriented diagnostic assays. The interaction between S protein and tissue-specific host cell receptors (ACE2) trigger fusion of viral envelop with cellular plasma or endosomal membrane, followed by a series of events relating to virus infection and host cell response.At present, no clearly defined treatment regimen and vaccines for SARS are available. The clinical presentation of SRAS is unspecific, resembling the symptoms of other forms of "atypical pneumonia". In this context, early and rapid identification of cases followed by appropriate management such as isolation of suspect and probable cases and management of their close contacts is the only effective way for the control of SARS spread. Suspect and probable cases of SARS should be subjected to laboratory testing for exclusion or confirmation of SARS infection. The availability of reliable and highly specific laboratory tests is of critical importance in the early and rapid diagnosis of SARS.This study integrated many up-to-date technologies such as PCR, in vitro cloning, ELISA, and fluorescence detection. Several original ideas such as "simple secondary structure" for the selection of target nucleotide sequence, "spatially compressed arrangement" for efficient FRET, and "anti-contamination reverse transcriptionsystem" were raised and applied in the design of the experiments, leading to 6 great innovations, including the obtaining of highly specific primers for SARS CoV, successful design of TaqMan-BC fluorescent probes, establishment of a highly efficient method for viral extraction, construction of pseudo-viral particles, introduction of RNase resistant internal control, and the establishment of RT/UNG anti-contamination system. Quantitative Fluorescent RT-PCR System for SARS CoV Detection, ELISA RT-PCR System for SARS Assay, and Double-antigen Sandwich ELISA Assay for SARS Diagnosis released from this study were licensed by SFDA. The main points of the results from this study are summarized below:1. With the idea of evaluating the simple secondary structure of nucleotide sequence, a novel TaqMan-BC fluorescence probe was invented and successfully applied in the fluorescence detection system, which can reduce the detection background by over 2 fold.2. An original RT/UNG combination was successfully established which can effectively eliminate contaminations which are frequently encountered in RT-PCR operations.3. A rapid and sensitive assay combining RT-PCR with rapid nucleotide hybridization was established.4. We are the first in the world that utilized synthesized vrial-like particles to serve as the internal control for SARS CoV detection. By use viral-like particles to monitor the entire detection process, false positive and false negative results could be eliminated. The viral-like particles, unlike inactivated SARS CoV or SARS CoV RNA samples which are liable to degeneration and potentially contagious, but being used in most of the assay systems, are safe, stable, and easy to handle.5. The N protein and M protein of SARS CoV were successfully cloned, expressed, purified, and utilized for the development of the double-antigen sandwich ELISA assay system. The sensitivity, specificity, stability, and reproducibility of the assay system we developed reach the standards for laboratory quality control.6. Monoclonal antibodies against M and N proteins were successfully produced. The proteins produced in vitro which are safe to human can be used as positive controls for the assay process.7. 402 samples from SARS patients and 2106 samples from healthy donors, Non-SARS patients were tested with the double-antigen sandwich ELIS A assay system. The percentage of reactive cases detected with the samples collected at the period of the disease course ( day 10 to 30) by this system is 87%. 2106 samples from healthy donors, Non-SARS patients were tested with this SARS detection system with negative results.8. The percentage of reactive cases detected with the 202 gargling liquid samples collected from SARS patients by PCR system is 56.4%. The other samples from healthy donors, Non-SARS patients were tested with PCR SARS detection system with negative results.Three detection methods for SARS detection have been established.