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Application of pregnancy test paper to on-site instant detection of hepatitis B virus drug-resistant mutant genes

A hepatitis B virus and drug-resistant mutation technology, which is applied in the direction of resistance to vector-borne diseases, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of gene chip method being expensive, complicated steps, time-consuming, etc. problem, to achieve the effect of specific and portable detection and intuitive results

Active Publication Date: 2021-02-23
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing detection methods for HBV drug-resistant mutation genes have many disadvantages, such as cumbersome sequencing technology, expensive and time-consuming; real-time fluorescent PCR technology has poor specificity and requires professional equipment, which cannot realize on-site instant diagnosis; gene chip method is expensive , the steps are complicated; although the restriction fragment length polymorphism analysis method is simple to operate, it has the phenomenon of non-specific cutting of homologs, the accuracy rate is low, and it is easy to cause missed detection and misjudgment.

Method used

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  • Application of pregnancy test paper to on-site instant detection of hepatitis B virus drug-resistant mutant genes
  • Application of pregnancy test paper to on-site instant detection of hepatitis B virus drug-resistant mutant genes
  • Application of pregnancy test paper to on-site instant detection of hepatitis B virus drug-resistant mutant genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Real-time detection of HBV drug-resistant mutant genes by LAMP-coupled strand-displacement probes to verify the effectiveness of LAMP amplification primers

[0071] experimental method:

[0072] 1. Prepare 25 μL reaction system, including external primer pair (SEQ ID NO:1 and SEQ ID NO:2), internal primer pair (SEQ ID NO:3 and SEQ ID NO:4), dNTP, betaine and buffer, The buffer is 1× isothermal amplification buffer, add 1 μL concentration of 0, 2copies / μL, 20copies / μL, 2×10 2 copies / μL, 2×10 3 copies / μL, 2×10 4 Copies / μL HBV mutant template (SEQ ID NO:5); after annealing, add fluorescent quenching strand displacement probes (SEQ ID NO:7 and SEQ ID NO:8), Bst 2.0 DNA polymerase; finally put into real-time In a fluorescent quantitative PCR instrument, react at 63°C for 90min.

[0073] 2. The LAMP amplification product was characterized by 1% agarose gel electrophoresis. Weigh 0.25g agarose and dissolve it in 25mL 1×TAE solution, heat in microwave until disso...

Embodiment 2 3

[0078] Example 2 Preparation and Stability Test of Three-Dimensional Large Volume DNA Molecules

[0079] experimental method:

[0080] 1. Prepare a 20 μL reaction solution system, including RCA template (SEQ ID NO:12), RCA primer (SEQ ID NO:13), T4 DNA ligase and buffer, the buffer is 1×DNA ligase buffer, react at room temperature 3h.

[0081] 2. Prepare a 20 μL reaction solution system, including phi29 DNA polymerase, dNTP, BSA and buffer, the buffer is 1×RCA amplification buffer, and mix with the above reaction solution, react at 30°C for 48 hours, and react at 75°C for 10 minutes , washed by centrifugation, and redispersed in 40 μL HO 2 O middle.

[0082] 3. Vacuum freeze-dry the reaction product, and disperse it in water after being stored at room temperature for different periods of time, and investigate its morphology change.

[0083] Experimental results:

[0084] Such as image 3 As shown, there is no obvious shape change after lyophilization and storage at room ...

Embodiment 3

[0085] Example 3 Concentration Gradient Detection of Pregnancy Test Paper on HBV Drug Resistance Mutant Gene LAMP Amplified Simulation Loop Sequence

[0086] experimental method:

[0087]1. Coupling hCG signaling molecules on probe P1 as a signal probe; hybridizing probe P1, probe P2 and probe P3 to generate a three-way probe; wherein, probe P2 and probe P3 are used as detection probes .

[0088] 2. Configure a 20 μL system, including 5 μL of the rolling circle amplification product (the three-dimensional large-volume DNA molecule obtained in Example 2) and 20 nM three-way probe, and react at room temperature for 1 h.

[0089] 3. Add 0, 0.5nM, 2nM, 5nM, 20nM, 50nM and other different concentrations of HBV drug-resistant mutant gene LAMP amplification simulation loop sequence (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., the HBV The sequence of the simulated circle amplified by LAMP of the drug-resistant mutant gene is the same as that of the single-stranded DNA...

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Abstract

The invention discloses application of pregnancy test paper to on-site instant detection of hepatitis B virus drug-resistant mutant genes, and relates to the field of HBV drug-resistant mutant gene detection. The detection method comprises the following steps of, carrying out loop-mediated isothermal amplification; designing three probes according to an LAMP amplification product; utilizing a rolling circle amplification reaction to obtain a three-dimensional large-volume DNA molecule; and coupling an hCG signal molecule at P1 as a signal probe, combining a three-way probe generated after P1,P2 and P3 are hybridized with the rolling circle amplification product, hybridizing a plurality of repeated units in the product with the tail end of P2, fixing the signal probe to prevent the signalprobe from entering the pregnancy test paper, adding the LAMP amplification product to be hybridized with P2 and P3, and therefore replacing the signal probe to enter the pregnancy test paper for color development, and realizing the full homogeneous signal open type POCT detection of the HBV drug-resistant mutant gene. The pregnancy test paper is simple and convenient to operate, low in cost, freeof complex instruments, high in sensitivity and good in specificity.

Description

technical field [0001] The invention relates to the technical field of hepatitis B virus drug-resistant mutation gene detection, in particular to the application of a pregnancy test paper in the on-site detection of hepatitis B virus drug-resistant mutation gene. Background technique [0002] Hepatitis B virus (HBV) is the main cause of the infectious disease hepatitis B and can cause cirrhosis and liver cancer. At present, nucleoside analogues are the most commonly used drugs in the clinical treatment of hepatitis B, but long-term use of antiviral drugs will induce drug-resistant mutations in HBV genes, which will seriously affect the therapeutic effect. The gold standard for the detection of HBV drug-resistant mutation genes is direct sequencing of PCR products. In addition, commonly used methods include real-time fluorescent PCR, pyrosequencing, gene chips, and restriction fragment length polymorphism analysis. [0003] Existing detection methods for HBV drug-resistant m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/706C12Q1/6844C12Q2600/156C12Q2531/119C12Q2565/625Y02A50/30
Inventor 杜衍齐丽娟杨媚婷
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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