Described herein are systems and methods for multiplexed analysis of two or more targets in a test sample including a first set of particles including a first set of target-specific reagents and a first optically detectable identifier capable of emitting a first wavelength indicative of a first target, and at least one second set of particles including a second set of target-specific reagents and a second optically detectable identifier capable of emitting a second wavelength indicative of a second target; and at least one optically detectable reporter probe capable of constitutively emitting a third wavelength in response to reaction of the first set of target-specific reagents with the first target in the test sample and/or reaction of the second set of target-specific reagents with the second target in the test sample, wherein the first wavelength, the second wavelength, and the third wavelength are optically discernable from one another.

Provided herein is a method for denaturation bubble-mediated target nucleic acid amplification and related kits and uses thereof. The method facilitates the generation of denaturation bubbles in a duplex target nucleic acid molecule through the application of swift temperature changes during a thermal cycle, thereby accelerating the strand exchange amplification (SEA) reaction. The kits comprise specially designed primers and polymerase configured for performing the method. The methods and kits disclosed herein can be used under various scenarios, such as diagnosis of infectious or genetic diseases, sample quality control, and single nucleotide polymorphism (SNP) profiling.

Provided herein is a method for denaturation bubble-mediated target nucleic acid amplification and related kits and uses thereof. The method facilitates the generation of denaturation bubbles in a duplex target nucleic acid molecule through the application of swift temperature changes during a thermal cycle, thereby accelerating the strand exchange amplification (SEA) reaction. The kits comprise specially designed primers and polymerase configured for performing the method. The methods and kits disclosed herein can be used under various scenarios, such as diagnosis of infectious or genetic diseases, sample quality control, and single nucleotide polymorphism (SNP) profiling.

The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.

The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.

A series of hybridisations is performed for forming a target double-stranded nucleic acid from initial fragments, where each further hybridisation step hybridises the direct products of a pair of earlier hybridisation steps. For at least one further hybridisation step H.sub.F, both of the corresponding pair of earlier hybridisation steps H.sub.E comprise an error-detecting type of hybridisation step, which includes an error detecting operation to detect whether the hybridised fragments formed in the error-detecting type of hybridisation step H.sub.E comprise at least one erroneous hybridised fragment, and discarding at least part of the erroneous fragment to exclude it from a subsequent further hybridisation step. By detecting and removing erroneous fragments throughout a staged and controlled hybridisation process, erroneous fragments are prevented from diluting the pool of error-free fragments at each hybridisation step, to improve yield.

A series of hybridisations is performed for forming a target double-stranded nucleic acid from initial fragments, where each further hybridisation step hybridises the direct products of a pair of earlier hybridisation steps. For at least one further hybridisation step H.sub.F, both of the corresponding pair of earlier hybridisation steps H.sub.E comprise an error-detecting type of hybridisation step, which includes an error detecting operation to detect whether the hybridised fragments formed in the error-detecting type of hybridisation step H.sub.E comprise at least one erroneous hybridised fragment, and discarding at least part of the erroneous fragment to exclude it from a subsequent further hybridisation step. By detecting and removing erroneous fragments throughout a staged and controlled hybridisation process, erroneous fragments are prevented from diluting the pool of error-free fragments at each hybridisation step, to improve yield.

Provided herein are methods and systems for detecting the presence or absence of multiple target nucleic acids in partitions of a digital amplification assay. In one embodiment of the method, different probes labeled with the same signal-generating label are distinguishable from each other as a result of having different melting temperatures in the presence of their cognate target nucleic acids. Following amplification of the target nucleic acids in a digital assay, signals are measured at three or more different temperatures and at least two relational values between signals measured at the three or more different temperatures are calculated and plotted against each other to classify partition subsets according to their target nucleic acid occupancy.

Provided herein are methods and systems for detecting the presence or absence of multiple target nucleic acids in partitions of a digital amplification assay. In one embodiment of the method, different probes labeled with the same signal-generating label are distinguishable from each other as a result of having different melting temperatures in the presence of their cognate target nucleic acids. Following amplification of the target nucleic acids in a digital assay, signals are measured at three or more different temperatures and at least two relational values between signals measured at the three or more different temperatures are calculated and plotted against each other to classify partition subsets according to their target nucleic acid occupancy.

Disclosed is a method of amplifying a nucleic acid sequence, wherein the method comprises subjecting a reaction mixture to at least one amplification cycle, wherein the reaction mixture comprises a double-stranded nucleic acid and at least two primers capable of annealing to complementary strands of the double-stranded nucleic acid and amplifying at least one short tandem repeat (STR) using a Family A DNA polymerase in a Fast PCR protocol having a two-step amplification cycle in 25 seconds or less. Also disclosed are real-time PCR methods using the two-step protocol and kits for STR profiling using the Fast PCR protocol.