Entropy-driven assisted T7 RNA polymerase amplification-activated CRISPR/Cas13a activity for SARS-CoV-2 detection in human pharyngeal swabs and environment by an electrochemiluminescence biosensor.

In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti3C2Tx-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.

Reverse Genetics of Avian Coronavirus Infectious Bronchitis Virus

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA;that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA dependant RNA polymerase. Copyright © Springer Science+Business Media New York 2016.