جستجوی مقالات مرتبط با کلیدواژه "rt-pcr optimization" در نشریات گروه "پزشکی"

Coronaviridae family cause respiratory diseases ranging from common cold to severe Respiratory diseases such as SARS, MERS and new emerging coronavirus disease COVID-19.  Also the family including four other human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) with flu likes symptoms. Coronaviruses cannot be distinguished clinically from other respiratory infectious agents. Based on the health importance and widespread distribution of respiratory infections, the current study was designed for diagnosis of Pancoronaviruses.  

Nasopharyngeal swabs from 200 patient suspected viral upper respiratory tract infection analyzed using optimized RT-PCR assay.  The constructive specific degenerate primers were used for amplification of rep1b ORF from coronaviruses genome. The 354bp DNA fragment related to 229E coronavirus polymerase gene was amplified from Amplirun Total Respiratory Viral Panel Control (Vircell) template by RT-PCR. Amplified product ligated into T-easy vector (Promega). Plasmid then transformed to Top 10 Fchr('39') strain by chemical method and Positive colonies were selected using colony PCR with gene specific primers. Diagnostic restriction enzyme digestion was done with EcoRI restriction enzyme. Vector was linearized by SacI restriction enzyme and In-vitro transcription was performed using TranscriptAid T7 High Yield Transcription Kit. DNA was removed with DNase I treatment. Then the detection limit of the specific Rep1b primers was determined by Two- Step RT-PCR with synthetic RNA concentration gradient. All of samples were negative for Pancoronavirus.

All of these samples were negative for Pancoronavirus.

Larger sample sizes and proper sampling procedure may improve the chances of viral RNA detection.