This disclosure provides methods and materials for determining whether a particular nucleic acid sequence is present in a test sample. Nucleic acid in the sample is treated with a nuclease mixture that generates monoribonucleotides if the target sequence is present. An exemplary nuclease mixture is a CRISPR associated protein that has endonuclease collateral activity (such as Cas12a), in combination with a guide RNA complementary to the target sequence, a reporter oligonucleotide, and an exonuclease. Upon binding of the Cas/gRNA complex to the target sequence in the sample, the endonuclease activity of the Cas protein cleaves the reporter oligonucleotide internally, rendering it suitable for digestion by the exonuclease. Monoribonucleotides that are generated as a consequence are measured by a bioluminescence detection means such as luciferase. The assay method is sufficiently robust, sensitive, and specific for use with a variety of biological test samples, sometimes without amplification.

This disclosure provides methods and materials for determining whether a particular nucleic acid sequence is present in a test sample. Nucleic acid in the sample is treated with a nuclease mixture that generates monoribonucleotides if the target sequence is present. An exemplary nuclease mixture is a CRISPR associated protein that has endonuclease collateral activity (such as Cas12a), in combination with a guide RNA complementary to the target sequence, a reporter oligonucleotide, and an exonuclease. Upon binding of the Cas/gRNA complex to the target sequence in the sample, the endonuclease activity of the Cas protein cleaves the reporter oligonucleotide internally, rendering it suitable for digestion by the exonuclease. Monoribonucleotides that are generated as a consequence are measured by a bioluminescence detection means such as luciferase. The assay method is sufficiently robust, sensitive, and specific for use with a variety of biological test samples, sometimes without amplification.

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule.

Methods for identifying co-occurrence of nucleic acid segments in a nucleic acid sample from a specimen including obtaining a nucleic acid sample from a specimen, determining sequences of first and second nucleic acid segments in nucleic acid fragments of the sample to generate a first and second sets of sequences, generating a first and second sets of probes from the first and second sets of sequences, exposing a detection sample to a member of the first set of probes and a member of the second set of probes, performing a hybridization analysis to determine whether the members of the first and second sets of probes hybridize to the detection sample, and determining whether the first and second nucleic acid segments co-occur in a common cell of the specimen.

Systems and methods for detection of a signal from droplets of an emulsion. An exemplary system may comprise a fluid transporter having a tube with an open end for aspirating droplets, a singulator to arrange the droplets in single file and to space the single-file droplets from one another, and a detection channel in optical communication with a detector configured to detect a signal from droplets. In some embodiments, the singulator may have a channel junction at which a stream of droplets in single file is combined with a stream of spacing fluid, and a tapered spacing channel extending downstream from the channel junction toward the detection channel. In some embodiments, the fluid transporter may suck droplet-containing fluid and spacing fluid through the detection channel from respective sources. In some embodiments, droplets may be subjected to a disaggregation routine before they are passed through the detection channel.

Systems and methods for detection of a signal from droplets of an emulsion. An exemplary system may comprise a fluid transporter having a tube with an open end for aspirating droplets, a singulator to arrange the droplets in single file and to space the single-file droplets from one another, and a detection channel in optical communication with a detector configured to detect a signal from droplets. In some embodiments, the singulator may have a channel junction at which a stream of droplets in single file is combined with a stream of spacing fluid, and a tapered spacing channel extending downstream from the channel junction toward the detection channel. In some embodiments, the fluid transporter may suck droplet-containing fluid and spacing fluid through the detection channel from respective sources. In some embodiments, droplets may be subjected to a disaggregation routine before they are passed through the detection channel.

Provided is a surface probe-based quantitative PCR testing system, comprising: (a) a solid phase carrier; (b) a specific primer pair of a sequence to be tested, which comprises a first primer and a second primer; and (c) a quenching probe. Further provided are a method for quantitative PCR testing and a kit.

Provided is a surface probe-based quantitative PCR testing system, comprising: (a) a solid phase carrier; (b) a specific primer pair of a sequence to be tested, which comprises a first primer and a second primer; and (c) a quenching probe. Further provided are a method for quantitative PCR testing and a kit.

Methods of sequencing molecules based on luminescence lifetimes and/or intensities are provided. In some aspects, methods of sequencing nucleic acids involve determining the luminescence lifetimes, and optionally luminescence intensities, of a series of luminescently labeled nucleotides incorporated during a nucleic acid sequencing reaction. In some aspects, the disclosure provides compositions comprising luminescently labeled nucleotides.

Methods of sequencing molecules based on luminescence lifetimes and/or intensities are provided. In some aspects, methods of sequencing nucleic acids involve determining the luminescence lifetimes, and optionally luminescence intensities, of a series of luminescently labeled nucleotides incorporated during a nucleic acid sequencing reaction. In some aspects, the disclosure provides compositions comprising luminescently labeled nucleotides.