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High-specificity nucleic acid detection reagent and using method thereof

A detection reagent and high-specificity technology, applied in the field of high-specificity nucleic acid detection reagents, can solve problems affecting the high specificity of nucleic acid, and achieve low-cost detection, easy operation, and high-fidelity specificity detection

Pending Publication Date: 2019-06-07
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods all need to perform PCR amplification on the nucleic acid to be tested first, and then use nuclease to hydrolyze the PCR amplification product into different fragments, thus affecting the high specificity of nucleic acid detection

Method used

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  • High-specificity nucleic acid detection reagent and using method thereof
  • High-specificity nucleic acid detection reagent and using method thereof
  • High-specificity nucleic acid detection reagent and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0035] Example The highly specific nucleic acid detection reagent combined with RCA technology detects let-7e, a member of the microRNA let-7 family.

[0036] MicroRNA let-7 is a 22-25 nucleotide long microRNA family with 9 members (let-7a~let-7i), they are involved in all stages of the life process and are important gene expression regulatory molecules one. However, family members differ very little (1-3 nucleotides), and accurate detection of family members is challenging. In this example, the microRNA family member let-7e is detected by rolling circle amplification technology (RCA for short) after the processed sample is detected by a highly specific nucleic acid detection reagent.

[0037] 1. Design specific probes and prepare samples.

[0038] Taking the microRNA let-7 family (let-7a ~ let-7f) as the detection object, DNA probe probe 7E was designed according to the nucleotide sequence of let-7 family members, as shown in Table 1.

[0039]

[0040] Nucleotides marke...

Embodiment 2

[0070] Example 2 Let-7e, a member of the microRNA let-7 family, was detected by combining the Lego-specific nucleic acid detection reagent with PCR technology.

[0071] In this example, samples 1 and 2 in Example 1 were first treated with highly specific nucleic acid detection reagents, and then let-7e was detected by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR for short). Samples not treated with CEL I served as the control group.

[0072] In the experiment, the Taqman probe RT-qPCR kit from Thermo-Fisher Scientific was used to detect the above samples.

[0073] 1. Process samples with highly specific nucleic acid detection reagents.

[0074] Formation of hybrid double strands: Mix 200 nM 5 μl of specific probe probe 7E with 100 nM 5 μl of sample 1 and 100 nM 5 μl of sample 2 respectively, raise the temperature to 95°C, and incubate for 5 minutes. Cool down to room temperature naturally.

[0075] Enzyme digestion reaction to remove non-ta...

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Abstract

The invention relates to high-specificity nucleic acid detection in the fields of basic research (gene expression regulation, biological evolution, species identification and genotyping) and application research (drug development, forensic identification, food and drug safety supervision, customs inspection and quarantine and the like), in particular to a high-specificity nucleic acid detection reagent and a using method thereof. The main components of the detection reagent include a specific probe for detecting a target nucleic acid strand and single-stranded nuclease capable of degrading single-stranded nucleic acid. The high-specificity nucleic acid detection reagent disclosed by the invention is simple and convenient to operate, fast and convenient, and high in cost performance, can meet the requirement of automation, is time-saving and labor-saving, and is convenient to realizing large-scale experiments and basic research.

Description

technical field [0001] The present invention relates to highly specific nucleic acid detection in the fields of basic research (gene expression regulation, biological evolution, species identification, genotyping) and applied research (drug development, forensic identification, food and drug safety supervision, customs inspection and quarantine, etc.), In particular, it relates to a highly specific nucleic acid detection reagent and a use method thereof. Background technique [0002] One of the important research contents of modern biology (botany, zoology, microbiology, marine biology, paleontology, paleoanthropology, etc.) is nucleic acid identification and nucleic acid detection. Complete genome information can enable humans to conduct in-depth, multi-perspective, and comprehensive analysis and interpretation of the origin, evolution, migration, and extinction of various extinct and existing species on the earth at the genetic level, because the genome Nucleotide sequenc...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6844C12Q1/6851
Inventor 张斌关一夫袁莹李硕
Owner 中国医科大学
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