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10.1186/s12985-020-01405-y http://scihub22266oqcxt.onion/10.1186/s12985-020-01405-y 32843049!7445803!32843049     free     free     free Warning: file_get_contents(https://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=32843049&cmd=llinks): Failed to open stream: HTTP request failed! 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A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E |pdf=|usr=}}{{32843049}} gab.com Text A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E JO: Virol J http://www.kidney.de/pget.php?&tw=1&pmid=32843049 #FOAvip BACKGROUND: Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. METHODS: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 x 10(6) to 1.2 x 10(1) copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 muL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. RESULTS: HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 x 10(3) viral copies/mL with 96.30% sensitivity. CONCLUSION: We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology. Twit Text FOAVip A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E ????Virol J http://www.kidney.de/pget.php?&tw=1&pmid=32843049 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=32843049 #FOAvip English Wikipedia <ref>{{Cite pmid|32843049}}</ref> A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E #MMPMID32843049 Morehouse ZP; Proctor CM; Ryan GL; Nash RJ Virol J 2020[Aug]; 17 (1): 129 PMID32843049show ga BACKGROUND: Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. METHODS: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 x 10(6) to 1.2 x 10(1) copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 muL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. RESULTS: HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 x 10(3) viral copies/mL with 96.30% sensitivity. CONCLUSION: We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology. |Cell Line[MESH] |Coronavirus 229E, Human/*genetics/isolation & purification[MESH] |Coronavirus Infections/*diagnosis[MESH] |Humans[MESH]A novel two-step, direct-to-PCR method for virus detection off swabs u(epmc).pdf DeepDyve Pubget Overpricing