The present disclosure generally relates to systems and methods for detecting viruses, e.g., using microfluidic devices. Certain embodiments are generally directed to systems and methods that are able to detect pathogens such as viruses or bacteria by encapsulating a sample in droplets, and applying amplification reagents to the droplets able to amplify nucleic acids therein, e.g., using loop mediated isothermal amplification (LAMP) or other amplification techniques. In addition, some aspects are generally directed to identifying a species in a sample, e.g., at very low concentrations. In some cases, the sample may be broken into droplets, arid the droplets determined to determine the species.

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.

A method of preparing a library of library constructs by multiplex amplification for use in targeted next generation sequencing is described. The method comprises the steps of: (a) providing a reaction vessel comprising (i) a plurality of different target sequences, (ii) a plurality of target capture primer pairs, and (iii) one or more tagging primer pairs, (b) performing sequential rounds of amplification at sequential annealing temperatures configured to amplify the target sequences, generate target sequences comprising first or second read sequences, and provide a reaction product comprising library constructs in a sequential manner; and (c) capture of the library of constructs from the reaction product. One of the forward and reverse tagging primers comprises a purification label at the 5′ end, and is provided at a limiting concentration whereby the library constructs comprises an abundance of partial constructs containing only one indexing sequences and only one adapter sequences, and a limited number of full (complete) constructs containing the first and second indexing sequences, the first and second adapter sequences and the purification label. The capture step comprises capturing the full (complete) constructs from the reaction product using the purification label.

A method of preparing a library of library constructs by multiplex amplification for use in targeted next generation sequencing is described. The method comprises the steps of: (a) providing a reaction vessel comprising (i) a plurality of different target sequences, (ii) a plurality of target capture primer pairs, and (iii) one or more tagging primer pairs, (b) performing sequential rounds of amplification at sequential annealing temperatures configured to amplify the target sequences, generate target sequences comprising first or second read sequences, and provide a reaction product comprising library constructs in a sequential manner; and (c) capture of the library of constructs from the reaction product. One of the forward and reverse tagging primers comprises a purification label at the 5′ end, and is provided at a limiting concentration whereby the library constructs comprises an abundance of partial constructs containing only one indexing sequences and only one adapter sequences, and a limited number of full (complete) constructs containing the first and second indexing sequences, the first and second adapter sequences and the purification label. The capture step comprises capturing the full (complete) constructs from the reaction product using the purification label.

A system for nucleic acid (NA) amplification includes a light source configured to emit a first excitation light based on a control signal, a reaction chamber configured to house a solution including a plurality of first nucleic acids (NAs), the plurality of first NAs being configured to amplify in response to the first excitation light, the solution being configured to emit a second light in response to heating by the first excitation light and to emit a third light in response to amplification of the plurality of first NAs, a detector configured to detect the second and third lights and to generate a temperature signal corresponding to the second light and a first fluorescence signal corresponding to the third light, and a lens module configured to focus the second and third lights onto the detector.

A system for nucleic acid (NA) amplification includes a light source configured to emit a first excitation light based on a control signal, a reaction chamber configured to house a solution including a plurality of first nucleic acids (NAs), the plurality of first NAs being configured to amplify in response to the first excitation light, the solution being configured to emit a second light in response to heating by the first excitation light and to emit a third light in response to amplification of the plurality of first NAs, a detector configured to detect the second and third lights and to generate a temperature signal corresponding to the second light and a first fluorescence signal corresponding to the third light, and a lens module configured to focus the second and third lights onto the detector.

This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an 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 also 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-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), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an 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 also 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-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), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

Provided herein are methods for de-crosslinking fixed biological samples (e.g., fixed biological samples including aminal crosslinks). The compositions and methods disclosed can de-crosslink oligonucleotides (e.g., DNA or RNA) or proteins from fixed biological samples (e.g., fixed biological samples with aminal crosslinks), wherein the de-crosslinked biological sample is compatible with and can be used in spatial gene expression analysis.