Abstract: A microfluidic assay device having a chemiluminescent testing assembly including a detection area, a sample inlet, and a microfluidic network including a first and second path extending to the detection area configured to capture a target analyte. When a fluid is provided to the sample inlet, a first portion of the fluid rehydrates a first dried reagent disposed along the first path to produce a first rehydrated reagent and a second portion of the fluid rehydrates a second dried reagent to produce a second rehydrated reagent. The first rehydrated reagent and the second rehydrated reagent are then sequentially delivered to the detection area by capillary-driven flow to perform the assay, where they react, optionally in the presence of a catalyst to form a chemiluminescent signal.

Abstract: Provided herein are methods and kits for discriminating a target sequence from a reference sequence in a polymerase chain reaction (PCR) by use of a promiscuity-blocking nucleotide juror (PBNJ) oligonucleotide.

Abstract: Provided are methods and kits for detecting and quantifying a sequence difference relative to a target polynucleotide sequence, and methods for developing assays. Target polynucleotide sequences are examined to determine if any of the sequences vary by at least one nucleotide difference. Promiscuous probes that have on and off-target binding at different binding efficiencies at permissive temperature are used, so that differences in polynucleotide sequences are reliably detected and quantified in a single well with fewer types of labeled probes, including by polymerase chain reaction of any of a range of sequences where there is interest in detecting an at least one nucleotide difference relative. The target sequences may be a reference polynucleotide sequence indicative of a first state, such as “normal” and another sequence that varies by at least one polynucleotide indicative of a different second state, such as a mutation, disease condition, or predisposition thereto.