Cloning Of SARS Coronavirus Full-length Genome And Development Of A Method For Concentrating And Purifying SARS Coronavirus RNA With Magnetic Beads Capture System

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, the SARS coronavirus (SARS-Cov). The disease is unusual in its high level of infectivitv and mortality. The epidemic in 2003 spread worldwide, affecting more than 8000 individuals and resulting in more than 900 deaths.SARS coronavirus is predominantly spread by droplet or by direct and indirect contact. Manipulations involving SARS coronavirus samples require BSL-3 facilities and BSL-3 work practices. These limit the acquirement of SARS research materials and baffle the expansion of research for SARS. The problems will be partially resolved by cloning of SARS coronavirus full-length genome and at the same time the genetic substances will be preserved for ever. In this study, 32 pairs of nested primers were designed initially according to the published sequences of SARS coronavirus gene. 32 target cDNA fragments were amplified from SARS coronavirus GDH strain with nested RT-PCR method, in which neighbour fragments overlapped about 300—500bp each other and then all fragments were cloned successfully. Clones were identified with sequencing. The results showed that the sequence of all the clones overlay the SARS coronavirus full-length genome and it was found to have a 99.9% match when compared with the published sequence of BJ01(AY278488).