Provided herein are methods and compositions for rapid, highly sensitive detection of SARS-CoV-2 in biological samples. In particular, provided herein is a rapid, low-cost method for detecting SARS-CoV-2 that provides reliable, visible test results and does not require PCR reagents, elaborate biosafety precautions, or sophisticated laboratory equipment.

Provided herein are methods and compositions for rapid, highly sensitive detection of SARS-CoV-2 in biological samples. In particular, provided herein is a rapid, low-cost method for detecting SARS-CoV-2 that provides reliable, visible test results and does not require PCR reagents, elaborate biosafety precautions, or sophisticated laboratory equipment.

Provided herein is a method for detecting the presence of a COVID-19 virus RNA or other pathogenic respiratory viruses, such as an influenza virus, or other RNA of interest in a sample. Nucleic acids are obtained from the sample and are used as a template in a combined isothermal reverse transcription, RNAse H and isothermal amplification reaction to generate single stranded RNA amplicons containing sequences complementary to fluorescent labeled detector probes. The single-stranded RNA amplicons hybridize to the detector probe and to hybridization probes with sequences complementary to a sequence determinant in the COVID-19 or other virus RNAs. The microarray is imaged to detect fluorescent signals thereby identifying the virus.

Provided herein is a method for detecting the presence of a COVID-19 virus RNA or other pathogenic respiratory viruses, such as an influenza virus, or other RNA of interest in a sample. Nucleic acids are obtained from the sample and are used as a template in a combined isothermal reverse transcription, RNAse H and isothermal amplification reaction to generate single stranded RNA amplicons containing sequences complementary to fluorescent labeled detector probes. The single-stranded RNA amplicons hybridize to the detector probe and to hybridization probes with sequences complementary to a sequence determinant in the COVID-19 or other virus RNAs. The microarray is imaged to detect fluorescent signals thereby identifying the virus.

This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

A method generates a marker in a biological sample including a plurality of cells by means of oligonucleotide constructs. The method includes introducing at least a plurality of first oligonucleotide constructs into the biological sample. The plurality of first oligonucleotide constructs comprise a first promoter, a first nucleic acid sequence encoding a first fluorescent protein, and a first photoremovable cage molecule. The method also includes exposing, in particular scanning, at least a first region of the biological sample with a first spatially constrained light beam to form uncaged first oligonucleotide constructs in order to enable synthesis of first fluorescent proteins from the first nucleic acid sequence and generate at least a part of the marker in the first region of the biological sample.

A method generates a marker in a biological sample including a plurality of cells by means of oligonucleotide constructs. The method includes introducing at least a plurality of first oligonucleotide constructs into the biological sample. The plurality of first oligonucleotide constructs comprise a first promoter, a first nucleic acid sequence encoding a first fluorescent protein, and a first photoremovable cage molecule. The method also includes exposing, in particular scanning, at least a first region of the biological sample with a first spatially constrained light beam to form uncaged first oligonucleotide constructs in order to enable synthesis of first fluorescent proteins from the first nucleic acid sequence and generate at least a part of the marker in the first region of the biological sample.

A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.

A method of nucleic acid analysis is described, the method including the steps of (a) providing a sample comprising a plurality of end-blocked polynucleotides derived from a biological source; (b) digesting at least some of the end-blocked polynucleotides with a nucleic acid-directed endonuclease that targets a sequence of interest to produce polynucleotide fragments that comprise the sequence of interest and a ligatable end generated by endonuclease cleavage; (c) ligating a moiety to the ligatable end to produce a moiety-target polynucleotide construct; and (d) detecting the moiety-target polynucleotide construct or a transcription or translation produce produced from the moiety-target polynucleotide construct using mass spectrometry. The moiety may be an adaptor sequence with a promoter for RNA polymerase. The moiety may be a chemical moiety that is highly amenable to flight and detection in a mass spectrometer.