[Identification and molecular cloning and sequence analysis of a novel coronavirus from patients with SARS by RT-PCR].
OBJECTIVE
To investigate the etiologic agents of the SARS and develop diagnostic method for this disease.


METHODS
Thirty-six nasopharyngeal aspirate specimens from 27 patients with SARS in Shenzhen were collected. The samples were aliquotted to three parts and subjected to molecular assays for human metapneumovirus, chlamydia and a novel coronavirus, which was reported recently to be the etiologic agent of SARS. Nested RT-PCR was used to amplify the RNA polymerase gene of the novel coronavirus and the PCR products were sequenced directly or after cloned to pMD18-T vector.


RESULTS
Human metapneumovirus and chlamydia genes were detected in none of the specimens using the RT-PCR and nested-PCR, respectively. The novel coronavirus gene were amplified in 6 of 36 specimens, the sequence analysis indicated that this novel coronavirus is unrelated to any other coronavirus reported previously. The nucleotide and deduced amino acid alignment between this coronavirus and others was not more than 40% and 70% to 82%, respectively, while the nucleotide sequence cloned from the 6 patients were identical.


CONCLUSIONS
The SARS patients in Shenzhen were infected with coronavirus and this novel coronavirus is associated with SARS. The sequence analysis indicated that the coronavirus from SARS patients in Shenzhen is the same as that identified from other areas such as Canada and Hong Kong. A specific diagnostic nested RT-PCR was developed to identify this novel coronavirus infection.
Zhou
Bo-ping
BP
Shenzhen Eastlake Hospital, Shenzhen 518020, China.
Chen
Xin-chun
XC
Wang
Huo-sheng
HS
Li
Mei-zhong
MZ
Hu
Yi-wen
YW
Du
Fan
F
Xu
Liu-mei
LM
Yang
Gui-lin
GL
D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008875:Middle Aged; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D045169:Severe Acute Respiratory Syndrome / Q000821:virology*
D004279:DNA, Viral; D012367:RNA, Viral