Described herein is an aqueous, miscible, multiphase, one-pot detection system including a first phase comprising a low density solution comprising a nucleic acid detection system; and a second phase in diffusive communication with the first phase, the second phase having a higher density than the first phase, and the second phase including a nucleic acid amplification system. Also included are multiwell plates and/or devices including the system and methods of detecting target nucleic acids.

Described herein is an aqueous, miscible, multiphase, one-pot detection system including a first phase comprising a low density solution comprising a nucleic acid detection system; and a second phase in diffusive communication with the first phase, the second phase having a higher density than the first phase, and the second phase including a nucleic acid amplification system. Also included are multiwell plates and/or devices including the system and methods of detecting target nucleic acids.

It is an object of the present disclosure to provide a method for reducing the influence of baseline disturbances in a method for detecting a target nucleic acid in dry blood filter paper by real-time PCR using a fluorescent dye, with a simple method. The present disclosure provides a method for detecting a target nucleic acid in dry blood filter paper by real-time PCR, the method including: (1) amplifying the target nucleic acid in the dry blood filter paper by applying thermal cycles to a sample solution containing a dry blood filter paper punch piece and a PCR reagent, wherein the PCR reagent includes a fluorescently labeled probe; (2) optically detecting the fluorescence intensity of the sample solution for each of the thermal cycles; and (3) performing quantitative analysis of the target nucleic acid using data after a predetermined number of cycles of the optically detected data.

It is an object of the present disclosure to provide a method for reducing the influence of baseline disturbances in a method for detecting a target nucleic acid in dry blood filter paper by real-time PCR using a fluorescent dye, with a simple method. The present disclosure provides a method for detecting a target nucleic acid in dry blood filter paper by real-time PCR, the method including: (1) amplifying the target nucleic acid in the dry blood filter paper by applying thermal cycles to a sample solution containing a dry blood filter paper punch piece and a PCR reagent, wherein the PCR reagent includes a fluorescently labeled probe; (2) optically detecting the fluorescence intensity of the sample solution for each of the thermal cycles; and (3) performing quantitative analysis of the target nucleic acid using data after a predetermined number of cycles of the optically detected data.

The present disclosure provides compositions and methods related to the detection of an analyte-of-interest. In particular, the present disclosure provides compositions and methods related to the detection and/or quantification of an analyte-of-interest using a signal detection component in combination with a signal amplification component. By combining these components in a modular format, cell-free synthetic gene circuits can be generated or improved to address a specific biological or biomedical diagnostic need.

The present disclosure provides compositions and methods related to the detection of an analyte-of-interest. In particular, the present disclosure provides compositions and methods related to the detection and/or quantification of an analyte-of-interest using a signal detection component in combination with a signal amplification component. By combining these components in a modular format, cell-free synthetic gene circuits can be generated or improved to address a specific biological or biomedical diagnostic need.

A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.

A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.

Provided herein is a lateral flow diagnostic device and methods of using thereof. The device comprises a substrate and a first end, wherein the first end comprises a sample loading portion. The first end may further comprise a first region loaded with a detectable ligand, a CRISPR effector system, a detection construct, a first test band comprising a biotin ligand, and a second test band comprising a capture molecule for the detectable ligand. The detection construct may comprise an RNA oligonucleotide, having a first molecule such as FITC on a first end and a second molecule such as FAM on a second end. Contacting the sample loading portion with a sample causes the sample to flow from the sample loading portion of the substrate towards the first and second capture regions, thereby generating a detectable signal, which may be indicative of a disease state.

Provided herein is a lateral flow diagnostic device and methods of using thereof. The device comprises a substrate and a first end, wherein the first end comprises a sample loading portion. The first end may further comprise a first region loaded with a detectable ligand, a CRISPR effector system, a detection construct, a first test band comprising a biotin ligand, and a second test band comprising a capture molecule for the detectable ligand. The detection construct may comprise an RNA oligonucleotide, having a first molecule such as FITC on a first end and a second molecule such as FAM on a second end. Contacting the sample loading portion with a sample causes the sample to flow from the sample loading portion of the substrate towards the first and second capture regions, thereby generating a detectable signal, which may be indicative of a disease state.