15 results about "Asymmetric PCR" patented technology

The invention discloses a reagent and a method for detecting low-frequency pancreatic cancer KRAS G12D gene mutation based on a Cas12a-Cas13a combined reaction system. The reagent comprises a single-stranded substrate DNA (Deoxyribonucleic Acid), a TRNA (Transcription Ribonucleic Acid), a 12CrRNA, a 13CrRNA, a 12Report and a 13Report; the single-stranded substrate DNA is derived from genome DNA of a to-be-detected sample, after the genome DNA of the to-be-detected sample is extracted, asymmetric PCR is carried out by utilizing a PCR primer pair, namely, the single-stranded substrate DNA is amplified and comprises wild type WT and mutant type MT; the mutant MT is completely matched with a recognition region of 12CrRNA, and a base mismatch exists between the mutant MT and a recognition region of 13CrRNA; a base mismatch exists between the wild type WT and a recognition region of 12CrRNA, and the wild type WT is completely matched with a recognition region of 13CrRNA; the TRNA is a pre-synthesized single-stranded RNA, has a base mismatch with a recognition region of the 12CrRNA, and is completely matched with a recognition region of the 13CrRNA; the 12Reporter is a DNA (Deoxyribose Nucleic Acid) probe of the FAM and BHQ double-standard genes, and the 13Reporter is an RNA (Ribonucleic Acid) probe of the FAM and BHQ double-standard genes. The nucleic acid cleavage activities of Cas12a and Cas13a are jointly utilized to realize multiple signal amplification, so that low-frequency detection of KRAS G12D mutation, which is simple to operate and low in cost, is realized.

The present invention discloses a methylation DNA sequence detection system based on covalent organic framework materials, its detection method and application. The detection system includes a covalent organic framework nanoprobe, a DNA sample processing unit and a detection unit. The detection process does not rely on composite materials and the preparation process is simple. The present invention uses an organic small molecule containing a triphenylamine structure as a monomer raw material, and prepares a covalent organic framework nanoprobe through a Schiff base reaction. The DNA is subjected to bisulfite treatment, asymmetric PCR and fluorescence labeling by the DNA sample processing unit to form a DNA to be detected. Then, the detection unit measures the fluorescence of the combination of the DNA sequence to be detected and the covalent organic framework nanoprobe, so as to realize the methylation detection of the DNA sequence. This system can efficiently and conveniently detect the methylation DNA sequence, and the operation process is simple and easy to implement.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

The invention discloses nucleic acid aptamers of five organophosphorus pesticides and application of the nucleic acid aptamers. The method comprises the following steps: firstly, preparing carboxyl-functionalized magnetic nanoparticles, incubating and combining amino-modified ssDNA complementary chains with the carboxyl-functionalized magnetic nanoparticles, incubating and combining an ssDNA library with the complementary chains, incubating and reacting mixed liquid of five pesticides as a target with the library, performing magnetic separation after the reaction is finished, taking supernate, and performing symmetric PCR amplification, after amplification is finished, a gel electrophoresis experiment is carried out to enrich double-stranded DNA, then recovery is carried out, asymmetric PCR amplification is carried out on the recovered double-stranded DNA to obtain a next-level library, and in the last round of screening process, a pesticide is adopted for positive screening to obtain a corresponding to-be-sequenced library. And finally obtaining nucleic acid aptamer sequences of five organophosphorus pesticides. The method disclosed by the invention can be used for efficiently and directionally screening the aptamer with high specificity for each pesticide.

This invention discloses a promoter specifically expressed in leaf veins, its expression vector, and its applications. Belonging to the fields of genetic engineering and biotechnology, this invention uses thermal asymmetric PCR and chromosome walking technology to clone a full-length 1772 bp promoter sequence from *Pteris vittata*. This promoter replaces the CaMV35S promoter on the vector pCAMBIA1381Z, thereby driving the expression of the downstream GUS reporter gene. The recombinant plant expression vector containing this specific expression promoter is transformed into tobacco to verify its function, confirming that this promoter can drive the GUS gene in tobacco leaves. This invention utilizes this promoter to construct a plant expression vector driving the expression of the *Pteris vittata* arsenite reverse transport protein PvACR3. Transforming this vector into tobacco confirms that this specific promoter driving PvACR3 expression can specifically increase the arsenic content in tobacco leaves. The promoter of this invention can be used to cultivate transgenic arsenic-accumulating engineered plants, promoting the development of phytoremediation technology.

The invention discloses a universal CRISPR / Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR associated 12a) system, which can realize visual rapid detection of acinetobacter baumannii drug resistance genes. The system is combined with an asymmetric PCR technology to amplify one housekeeping gene and four drug-resistant genes of bacteria to generate a single-stranded DNA product, so that the amplification efficiency and the signal intensity are remarkably improved, and a high-specificity substrate is provided for subsequent reaction. The single-chain amplicon and the modular ring engineering hairpin are subjected to a strand displacement reaction, and Cas12a protein activation is accurately regulated and controlled; the activated Cas12a cuts the reporter molecule, and the product presents a visual visual signal through a lateral flow chromatography test strip. The strategy has prominent universality, crRNA does not need to be redesigned or an activating agent does not need to be added when five targets are detected, plug-and-play can be achieved only by replacing the hairpin response sequence, and the method can be conveniently expanded to detection of other bacteria, viruses and drug-resistant genes. The system is easy and convenient to operate, high in specificity and good in stability, provides a simplified and universal CRISPR diagnosis new scheme for clinical bacterial genotyping, and has important clinical application and popularization value.

The invention discloses a PCR-CRISPR method and a PCR-CRISPR system for integrated detection of drug-resistant sites of mycobacterium tuberculosis and rifampicin. Specifically, the invention discloses a PCR-CRISPR method for in-vitro non-diagnostic and non-therapeutic integrated detection of mycobacterium tuberculosis and rifampicin drug-resistant mutation sites, and the method comprises the following steps: (s1) providing a sample to be detected, and extracting total DNA; (s2) pushing nucleic acid liquid containing the total DNA into a first reaction chamber, and carrying out asymmetric PCR reaction to obtain a first fluorescence signal; (s3) pushing a PCR amplification product in the step (s2) into a second reaction chamber, carrying out CRISPR reaction in the second reaction chamber, and obtaining a second fluorescence signal for judging whether the target sequence exists or not; and (s4) combining and analyzing the first fluorescence signal and the second fluorescence signal to obtain a detection result of the mycobacterium tuberculosis and rifampicin drug-resistant mutation site. The method disclosed by the invention is high in detection sensitivity, convenient to operate and low in cost, and can be used for effectively screening tuberculosis and multidrug-resistant tuberculosis.

The present application relates to the technical field of in vitro detection, in particular to primer probe combination, reagent and application, and provides primer probe combination, reagent or kit for detecting HPV DNA and mRNA, which aims at simply, quickly and accurately detecting multiple types of HPV DNA and mRNA in a single reaction to achieve the purpose of typing.The present application uses asymmetric PCR technology-melting curve technology to perform fluorescent PCR detection and typing on the DNA or mRNA fragments of the 9 types of HPV 16, 18, 31, 33, 45, 52, 58, 59 and 68 in a single tube.

The application discloses an ABO genotyping composition and kit based on a multiple asymmetric PCR probe melting curve and application. The application can realize accurate ABO genotyping in one batch of PCR reactions, and the whole detection process is simple, rapid, accurate and efficient, and has high practical value. The application makes up for the shortcomings of the existing serological detection method, has the advantages of standardization, low cost and low technical barrier for clinical popularization, and has a wide application prospect in ABO genotyping for Chinese population.

The invention provides a molecular comparator based on a miRNA pair and application of the molecular comparator. The molecular comparator comprises a miRNA marker, an asymmetric PCR reaction device, a TMSD reaction device and a fluorescence signal output device, the method comprises the following steps: sequentially passing miRNA markers through an asymmetric PCR reaction device to obtain ssDNA, placing the ssDNA in a TMSD reaction device to obtain a probe E and a probe F, hybridizing the probe E and the probe F, and carrying out threshold-based comparison on the relative abundance of miR-148a-3p and miR-30d-5p on a molecular level by using a fluorescence signal output device; the relative expression level of the specific miRNA pair is internally normalized, interference of irrelevant physiological changes is reduced, and compared with diagnosis based on the absolute content of a single miRNA, the method has more excellent performance and can be applied to preparation of a non-small cell lung cancer detection kit.

The disclosure provides an asymmetric PCR amplification method for preparation of single-stranded product and primers and kits useful therefor.

The present invention discloses an application of the melting curve method in the genotyping and qualitative analysis of human papillomavirus. In the present invention, specific primers and corresponding specific fluorescent probes are designed for the E6 / E7 DNA of 18 high-risk and intermediate-risk types of HPV and the internal reference β-globin, and multiple targets in a reaction can be simultaneously detected by using asymmetric PCR amplification combined with the melting curve technology. The present invention can perform qualitative and genotyping detection of 18 types of human papillomavirus. The 18 types of HPV in the present invention refer to HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The present invention uses the carcinogenic gene E6 / E7 DNA of HPV as the detection target, which can avoid the missed detection caused by using HPV L1 DNA as the detection target. At the same time, the melting curve technology used in the present invention has the advantages of high detection sensitivity, good specificity, and high throughput.

The invention relates to an oligonucleotide combination and kit for detecting and distinguishing cryptococcus. The oligonucleotide combination comprises a specific primer, a guide chain and a reporter probe, wherein the specific primer, the guide chain and the reporter probe are designed aiming at a cryptococcus neoformans CAP59 gene and a cryptococcus gattii GPA1 gene. The kit comprises the oligonucleotide combination, pfAgo enzyme, divalent manganese ions and a reaction buffer solution, and can be used for non-diagnostic purpose detection. The method comprises the following steps: carrying out asymmetric PCR amplification on a sample DNA to obtain a single-chain product, incubating the single-chain product with pfAgo enzyme, a guide chain and a reporter probe together to carry out a cleavage reaction, and finally distinguishing the cryptococcus species through fluorescence signals (such as FAM and ROX). The method has the advantages of high specificity and sensitivity (the lower detection limit reaches 1CFU / mL), no cross reaction, short detection time (about 60 minutes), accurate verification in clinical cerebrospinal fluid samples, and suitableness for rapid identification of cryptococcus.

The present application relates to the application of graphene oxide in the preparation of single-stranded DNA secondary library for aptamer screening. Specifically, graphene oxide is incubated with the product after asymmetric PCR amplification, and the single-stranded DNA secondary library on graphene oxide can be obtained after centrifugation and washing. The method of the present application is simple, does not affect the specificity and affinity of aptamer, has the advantages of low cost and high efficiency, overcomes the problems of existing single-stranded DNA secondary library preparation methods, such as many steps, high operation requirements and high cost, and helps to screen high-quality aptamer.