A microfluidic device for amplifying a nucleic acid includes a cartridge and a control part. The cartridge includes a tank part and a plurality of first chambers. The control part is configured to control execution of a thermal cycle, count a number of repetitions of the thermal cycle for each of the first chambers and store a count value, acquire a fluorescence intensity of each of the first chambers for each thermal cycle, and reset the count value of a defective chamber of which the fluorescence intensity is not within a predetermined range, discharge the solution from the defective chamber, and fill the defective chamber with a new solution from the tank part.

The invention relates to a method for detecting cell death using PCR (polymerase chain reaction) techniques, including qPCR/quantitative PCR or ddPCR/digital droplet PCR, or any other technique for detecting a small amount of DNA (such as nanostrings).

The invention relates to a method for detecting cell death using PCR (polymerase chain reaction) techniques, including qPCR/quantitative PCR or ddPCR/digital droplet PCR, or any other technique for detecting a small amount of DNA (such as nanostrings).

Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells, contamination dead cells, cell debris, and biofilm using two separation steps, either by centrifugation or filtration, performed in sequentially. Also provided is a method for isolating nucleic acids from intact cells using a first separation step followed by treatment with a nuclease and then a second separating step. Provided herein is a related method for isolating DNA from intact cells using a nuclease that produces DNA cuts on double stranded DNA, followed by a second separating step.

A method comprises (a) providing single-stranded DNA; (b) ligating a first adapter to a 3′ end of the single-stranded DNA to form a once adapter ligated nucleic acid strand, the first adapter having a first protruding random sequence that is at least 3 bases long and that acts as a splint to join the single-stranded DNA with the first adapter; (c) ligating a second adapter to a 5′ end of the once adapter ligated nucleic acid strand to form a twice ligated nucleic acid strand, the second adapter having a second protruding random sequence that is at least 3 bases long and that acts as a splint to join the once adapter ligated nucleic acid strand with the second adapter; and (d) performing an amplification reaction on the twice ligated nucleic acid strand, thereby generating copies of the twice ligated nucleic acid strand.

A method comprises (a) providing single-stranded DNA; (b) ligating a first adapter to a 3′ end of the single-stranded DNA to form a once adapter ligated nucleic acid strand, the first adapter having a first protruding random sequence that is at least 3 bases long and that acts as a splint to join the single-stranded DNA with the first adapter; (c) ligating a second adapter to a 5′ end of the once adapter ligated nucleic acid strand to form a twice ligated nucleic acid strand, the second adapter having a second protruding random sequence that is at least 3 bases long and that acts as a splint to join the once adapter ligated nucleic acid strand with the second adapter; and (d) performing an amplification reaction on the twice ligated nucleic acid strand, thereby generating copies of the twice ligated nucleic acid strand.

Methods according to aspects of the disclosure, compositions and kits therefore, include at least one, two, or three sets of amplification primers and hydrolysis probes with at least two separate corresponding readouts per set. According to aspects of the present disclosure, the at least two hydrolysis probes and associated pair of primers of each set are directed to opposite strands of an amplification product of the set. According to aspects of the present disclosure, one of the two hydrolysis probes in each set is directed to a first strand of the amplification product and therefore has a sequence complementary to the first strand of the amplification product and the second of the two hydrolysis probes in the set is directed to the second strand of the amplification product and therefore has a sequence complementary to the second strand of the amplification product.

Methods according to aspects of the disclosure, compositions and kits therefore, include at least one, two, or three sets of amplification primers and hydrolysis probes with at least two separate corresponding readouts per set. According to aspects of the present disclosure, the at least two hydrolysis probes and associated pair of primers of each set are directed to opposite strands of an amplification product of the set. According to aspects of the present disclosure, one of the two hydrolysis probes in each set is directed to a first strand of the amplification product and therefore has a sequence complementary to the first strand of the amplification product and the second of the two hydrolysis probes in the set is directed to the second strand of the amplification product and therefore has a sequence complementary to the second strand of the amplification product.

This application describes a fluorogenic nucleic acid kit or composition for quantitative detection of a target ribonucleic acid (RNA) sequence in a test sample comprising at least one pair of oligonucleotide probes comprising a photocatalyst probe and a profluorophore probe, wherein one of the photocatalyst probe and the profluorophore probe is complementary to and capable of specifically binding an upstream portion of the target RNA sequence, and the other probe is complementary to and capable of specifically binding to a downstream portion of the target RNA sequence, the photocatalyst probe comprises a first oligonucleotide covalently bound to a photocatalyst, the profluorophore probe comprises a second oligonucleotide covalently bound to a profluorophore, and the photocatalyst is activatable by exposure to light and a reducing agent to form a reduced, activated photocatalyst that, when both probes of the pair are hybridized to the target RNA sequence, is capable of photoreducing the profluorophore to form a detectable fluorophore. The application also describes methods for quantitative detection of target RNA.

This application describes a fluorogenic nucleic acid kit or composition for quantitative detection of a target ribonucleic acid (RNA) sequence in a test sample comprising at least one pair of oligonucleotide probes comprising a photocatalyst probe and a profluorophore probe, wherein one of the photocatalyst probe and the profluorophore probe is complementary to and capable of specifically binding an upstream portion of the target RNA sequence, and the other probe is complementary to and capable of specifically binding to a downstream portion of the target RNA sequence, the photocatalyst probe comprises a first oligonucleotide covalently bound to a photocatalyst, the profluorophore probe comprises a second oligonucleotide covalently bound to a profluorophore, and the photocatalyst is activatable by exposure to light and a reducing agent to form a reduced, activated photocatalyst that, when both probes of the pair are hybridized to the target RNA sequence, is capable of photoreducing the profluorophore to form a detectable fluorophore. The application also describes methods for quantitative detection of target RNA.