Browsing by Subject "phage phi 6"

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  • Gregorova, Pavlina; Heinonen, Minna-Maria Kristiina; Sarin, Peter (2022)
    Reverse transcription quantitative PCR (RT-qPCR) has emerged as the gold standard for virus detection and quantification, being utilized in numerous diagnostic and research applications. However, the direct detection of viruses has so far posed a challenge as the viral genome is often encapsidated by a proteinaceous layer surrounded by a lipid envelope. This necessitates an additional and undesired RNA extraction step prior to RT-qPCR amplification. To circumvent this limitation, we have developed a direct RT-qPCR method for the detection of RNA viruses. In our method, we provide a proof-of-concept using phage phi6, a safe-to-use proxy for pathogenic enveloped RNA viruses that is commonly utilized in e.g. aerosolization studies. First, the phage phi6 envelope is removed by 1% chloroform treatment and the virus is then directly quantified by RT-qPCR. To identify false negative results, firefly luciferase is included as a synthetic external control. Thanks to the duplex format, our direct RT-qPCR method reduces the reagents needed and provides an easy to implement and broadly applicable, fast, and cost-effective tool for the quantitative analysis of enveloped RNA viruses.center dot One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation.center dot Reduced reaction volume for sustainable and cost-effective analysis.(c) 2022 The Author(s). Published by Elsevier B.V.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
  • Hanhijarvi, Kalle J.; Ziedaite, Gabija; Bamford, Dennis H.; Haeggstrom, Edward; Poranen, Minna M. (2017)
    Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage phi 6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative strands are synthesized. We present single-molecule measurements of the viral ssRNA packaging by the phi 6 PC. Our data show that packaging proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Although the mean packaging velocity was relatively low (0.07-0.54 nm/sec), packaging could reach 4.62 nm/sec during the fast packaging phase.