A method of detecting a target sequence is provided, the method comprising: hybridizing an oligonucleotide probe with a nucleic acid present in a test sample, wherein the oligonucleotide probe has a hybridization sequence with respect to a target sequence of the nucleic acid and has at least one covalent bonding group that is crosslinkable with a target base in the target sequence by light irradiation, thereby forming an authentic hybridization product; irradiating light to the test sample after the hybridizing, wherein the covalent bonding group and the target sequence are crosslinked, thereby forming the authentic cross-liked hybridization product having a crosslinked structure; and denaturating the test sample by applying a denaturating condition in which the authentic crosslinked hybridization product is able to be maintained and a non-specific hybridization product is able to be dissociated and separating the oligonucleotide probe derived from the non-specific hybridization product.

A method of detecting a target sequence is provided, the method comprising: hybridizing an oligonucleotide probe with a nucleic acid present in a test sample, wherein the oligonucleotide probe has a hybridization sequence with respect to a target sequence of the nucleic acid and has at least one covalent bonding group that is crosslinkable with a target base in the target sequence by light irradiation, thereby forming an authentic hybridization product; irradiating light to the test sample after the hybridizing, wherein the covalent bonding group and the target sequence are crosslinked, thereby forming the authentic cross-liked hybridization product having a crosslinked structure; and denaturating the test sample by applying a denaturating condition in which the authentic crosslinked hybridization product is able to be maintained and a non-specific hybridization product is able to be dissociated and separating the oligonucleotide probe derived from the non-specific hybridization product.

The present disclosure is drawn to loop-mediated isothermal amplification (LAMP) reaction assemblies including a substantially hygroscopic agent free LAMP reagent mixture in combination with a solid-phase reaction medium. The present disclosure also includes systems for a chromatic LAMP analysis including a substantially non-reactive solid phase reaction medium, and a non-interfering reagent mixture. The present disclosure also includes solid phase LAMP reaction mediums comprising a substrate, an adhesive layer disposed on the substrate, a reaction layer disposed on the adhesive layer, and a spreading layer disposed on the reaction layer. The present disclosure also includes methods of testing for a presence of a target nucleotide sequence including providing a biological sample, and dispensing the sample into a test environment having a solid phase reaction medium in combination with a LAMP reagent mixture and a pH sensitive dye.

The present disclosure is drawn to loop-mediated isothermal amplification (LAMP) reaction assemblies including a substantially hygroscopic agent free LAMP reagent mixture in combination with a solid-phase reaction medium. The present disclosure also includes systems for a chromatic LAMP analysis including a substantially non-reactive solid phase reaction medium, and a non-interfering reagent mixture. The present disclosure also includes solid phase LAMP reaction mediums comprising a substrate, an adhesive layer disposed on the substrate, a reaction layer disposed on the adhesive layer, and a spreading layer disposed on the reaction layer. The present disclosure also includes methods of testing for a presence of a target nucleotide sequence including providing a biological sample, and dispensing the sample into a test environment having a solid phase reaction medium in combination with a LAMP reagent mixture and a pH sensitive dye.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

A direct chemical lysis composition includes an assay compatible buffer composition and an assay compatible surfactant. When combined with a specimen storage composition, such compositions prevent undesired modifications to nucleic acid and proteins lysed from cells in the biological sample. Assays of samples from such compositions do not require expensive and time-consuming steps such as centrifugation and prolonged high temperature processing. The direct chemical lysis composition of the present invention permits direct nucleic acid extraction from the cells in the biological sample without the need to decant off the transport media or otherwise exchange the transport media with assay compatible buffers. There is no need to combine the sample with proteinase K or another enzyme to extract nucleic acids from the cells. A method for lysing cells to obtain target nucleic acid for assay and a kit for combining the direct chemical lysis composition with a sample are also contemplated.

A direct chemical lysis composition includes an assay compatible buffer composition and an assay compatible surfactant. When combined with a specimen storage composition, such compositions prevent undesired modifications to nucleic acid and proteins lysed from cells in the biological sample. Assays of samples from such compositions do not require expensive and time-consuming steps such as centrifugation and prolonged high temperature processing. The direct chemical lysis composition of the present invention permits direct nucleic acid extraction from the cells in the biological sample without the need to decant off the transport media or otherwise exchange the transport media with assay compatible buffers. There is no need to combine the sample with proteinase K or another enzyme to extract nucleic acids from the cells. A method for lysing cells to obtain target nucleic acid for assay and a kit for combining the direct chemical lysis composition with a sample are also contemplated.

Provided herein includes a method for characterizing a target cell-free nucleic acid (cfNA) molecule present in a biological sample such as a urine sample. It comprises isolating total cfNAs from the biological sample without prior preprocessing such as centrifugation to remove cell debris, and characterizing the target cfNA molecule based on the isolated total cfNAs. When the target cfNA is a low molecular weight (LMW) molecule, the method additionally comprises a fractionation step to obtain LMW nucleic acids from the total cfNAs before characterization. The method can detect significantly more copies of the target cfNA molecule compared with existing methods which typically discard the cell debris from the biological sample. Another method is also provided, which substantially recovers cfNAs from the usually discarded cell debris, thus also capable of detecting significantly more copies of the target cfNA molecule.

Provided herein includes a method for characterizing a target cell-free nucleic acid (cfNA) molecule present in a biological sample such as a urine sample. It comprises isolating total cfNAs from the biological sample without prior preprocessing such as centrifugation to remove cell debris, and characterizing the target cfNA molecule based on the isolated total cfNAs. When the target cfNA is a low molecular weight (LMW) molecule, the method additionally comprises a fractionation step to obtain LMW nucleic acids from the total cfNAs before characterization. The method can detect significantly more copies of the target cfNA molecule compared with existing methods which typically discard the cell debris from the biological sample. Another method is also provided, which substantially recovers cfNAs from the usually discarded cell debris, thus also capable of detecting significantly more copies of the target cfNA molecule.