Nucleic acid probe composition, pretreatment liquid, nucleic acid detection kit and detection method

A technology of nucleic acid probe and pretreatment solution, which is applied in the field of nucleic acid probe composition, can solve problems such as inability to apply, and achieve the effect of reducing detection sensitivity and false positives

Pending Publication Date: 2022-05-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still relies on nucleic acid amplification technology, which requires professional equipment and personnel to operate, and cannot be applied to resource-scarce areas or large-scale rapid detection and other application scenarios

Method used

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  • Nucleic acid probe composition, pretreatment liquid, nucleic acid detection kit and detection method
  • Nucleic acid probe composition, pretreatment liquid, nucleic acid detection kit and detection method
  • Nucleic acid probe composition, pretreatment liquid, nucleic acid detection kit and detection method

Examples

Experimental program
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Effect test

preparation example Construction

[0092] In some embodiments, the preparation method of the connection pad includes the steps of: soaking the connection pad in a buffer solution containing bovine serum albumin and disodium hydrogen phosphate for 1-1000 minutes, then drying and storing in a desiccator. The marker-labeled reporter probe was sprayed on the ligation pad and stored in a dry place at room temperature.

[0093] In some embodiments, the preparation method of the reaction membrane includes the following steps: modifying the detection probe and the quality control probe respectively on the reaction membrane substrate to form a detection line (T line) and a quality control line (C line), placing them on the substrate containing the Skim milk was soaked in a triethanolamine-buffered saline solution, then dried at room temperature and stored in a desiccator.

[0094] In some embodiments, streptavidin is applied to the reaction membrane substrate. The 5' end of the detection probe is modified with biotin. ...

Embodiment 1

[0116] Example 1 (taking SARS-CoV-2 as an example)

[0117] see figure 1 and figure 2 , a nucleic acid detection device and a detection method thereof, the detection method adopts nucleic acid detection test strip 1 as a reaction unit, nucleic acid detection test strip 1 is placed in a cartridge 2, and the cartridge 2 has a push-pull structure 3. The nucleic acid detection test strip 1 used comprises a bottom plate 4, a sample pad 5, a connection pad 6, a nitrocellulose membrane and a water absorption pad 10 which are overlapped and fixed on the bottom plate 4 in sequence;

[0118] The connecting pad 6 is fixed with a reporter group, and the reporter group is colloidal gold labeled with probes; the base film of the reaction membrane 7 is a nitrocellulose membrane, on which a detection line 8 and a quality control line 9 are arranged; The detection line 8 is fixed with a detection probe; the quality control line 9 is fixed with a quality control probe.

[0119] The 5' end o...

Embodiment 2

[0145] Example 2 (taking SARS-CoV-2 as an example)

[0146] This example is the same as the preparation method in Example 1, except that the reporter group immobilized on the connection pad 6 is colloidal gold co-labeled with the reporter probe and horseradish peroxidase.

[0147] The specific detection method is:

[0148] Disperse 40 μl of the sample to be tested with a concentration of 10 pmol in 60 μl of sodium citrate buffer containing 10 nmol of complementary primers of labeled auxiliary regions, primary signal amplification chain and secondary signal amplification chain to obtain the test solution;

[0149] Add dropwise to sample pad 5 to be tested, drop 60 μl of sodium citrate buffer on sample pad 5 after 5-30 min, drop 60 μl of chromogenic substrate on sample pad 5 after 1-15 min, 1- After 30min, the color development result of the test strip was obtained;

[0150] Through naked eye detection, both the detection line 8 and the quality control line 9 are dark brown, a...

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Abstract

The nucleic acid probe composition comprises a detection probe, a quality control probe, a report probe, a first nucleic acid probe, a second nucleic acid probe and a signal amplification probe, the first nucleic acid probe comprises a first sequence region and a second sequence region, and the first sequence region is complementarily paired with a marker auxiliary sequence region of nucleic acid to be detected; the second nucleic acid probe comprises a third sequence region and a fourth sequence region, the third sequence region is complementarily paired with a capture auxiliary sequence region of nucleic acid to be detected, and the fourth sequence region is complementarily paired with the detection probe; the report probe and the quality control probe are complementarily paired; the signal amplification probe comprises an amplification chain front guide sequence region and a plurality of amplification sequence regions which are sequentially connected, the amplification sequence regions are complementarily paired with the reporter probe, and the amplification chain front guide sequence region is complementarily paired with the second sequence region of the first nucleic acid probe. The invention also discloses a nucleic acid detection method and a kit.

Description

technical field [0001] The present invention relates to the technical field of biochemical detection, in particular to a nucleic acid probe composition, a pretreatment solution, a nucleic acid detection kit and a detection method. Background technique [0002] Nucleic acid detection is the detection of DNA and RNA. It is the most advanced and reliable detection method for viruses and bacteria. It is commonly used in the detection of hepatitis B, hepatitis C, AIDS, respiratory pathogens, etc. At present, nucleic acid detection mainly adopts nucleic acid amplification technologies such as polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), and loop-mediated isothermal amplification (LAMP). Controlling the amplification of target nucleic acid fragments for detection has the advantages of improving detection specificity and reducing the probability of false positives. [0003] Chinese patent CN111593145A invented a CRISPR / Cas12 one-step n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6834C12N15/11
CPCC12Q1/6834C12Q2565/625C12Q2565/519C12Q2527/125
Inventor 王晗孙凯
Owner TSINGHUA UNIV
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