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Application of pregnancy test strips in on-site point-of-care detection of hepatitis B virus drug-resistant mutant genes

A hepatitis B virus and drug-resistant mutation technology, applied in the direction of resistance to vector-borne diseases, microbial measurement/testing, biochemical equipment and methods, etc., can solve the problem of expensive gene chip method, non-specific cutting of homologues, Missed detection, misjudgment and other problems, to achieve the effect of specific and portable detection

Active Publication Date: 2022-08-09
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • 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 strips in on-site point-of-care detection of hepatitis B virus drug-resistant mutant genes
  • Application of pregnancy test strips in on-site point-of-care detection of hepatitis B virus drug-resistant mutant genes
  • Application of pregnancy test strips in on-site point-of-care 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 resistance mutant genes with LAMP-coupled strand displacement probes to verify the effectiveness of LAMP amplification primers

[0071] experimental method:

[0072] 1. Prepare a 25 μL reaction system, including an outer primer pair (SEQ ID NO: 1 and SEQ ID NO: 2), an inner 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 of 0, 2 copies / μL, 20 copies / μL, 2×10 2 copies / μL, 2×10 3 copies / μL, 2×10 4 copies / μL of HBV mutant template (SEQ ID NO: 5); after annealing, fluorescent quenched strand displacement probes (SEQ ID NO: 7 and SEQ ID NO: 8), Bst 2.0 DNA polymerase were added; In a fluorescence quantitative PCR instrument, the reaction was performed at 63 °C for 90 min.

[0073] 2. The LAMP amplification products were characterized by 1% agarose gel electrophoresis. Weigh 0.25g of agarose and dissolve it in 25mL of 1×TAE solution, heat it in a mi...

Embodiment 2 3

[0078] Example 2 Preparation and stability test of three-dimensional bulk DNA molecules

[0079] experimental method:

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

[0081] 2. Configure 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-reacted solution, react at 30°C for 48 hours, and react at 75°C for 10 minutes , washed by centrifugation, and redispersed in 40 μL of H 2 O in.

[0082] 3. The reaction product was lyophilized in vacuum, stored at room temperature for different times, and then dispersed in water to investigate the change of its morphology.

[0083] Experimental results:

[0084] like image 3 As shown, no obvious morphological changes were observed after lyophilization and...

Embodiment 3

[0085] Example 3 Concentration gradient detection of HBV drug resistance mutant gene LAMP amplification simulation loop sequence by pregnancy test paper

[0086] experimental method:

[0087]1. Conjugate hCG signal molecule on probe P1 as a signal probe; hybridize 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 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 resistance mutant gene LAMP amplification simulation loop sequence (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., this HBV The sequence of the LAMP amplification simulation loop of the drug-resistant mutant gene was the same as that of the single-stranded DNA loop ...

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Abstract

The application of pregnancy test strips in the field point-of-care detection of hepatitis B virus drug resistance mutation genes relates to the field of HBV drug resistance mutation gene detection. The detection method includes: loop-mediated isothermal amplification; three probes are designed according to LAMP amplification products; three-dimensional bulk DNA molecules are obtained by using rolling circle amplification reaction; P1 is coupled with hCG signal molecule as signal probe, P1, P2 The three-way probe generated after hybridization with P3 is combined with the rolling circle amplification product. Multiple repeating units in the product are hybridized with the end of P2. The signal probe is fixed to prevent it from entering the pregnancy test paper, and the LAMP amplification product is added to make it bind to P2. , P3 hybridization to replace the signal probe into the pregnancy test strip for color development, and realize the full homogeneous signal open-type POCT detection of HBV drug resistance mutation genes. The invention has the advantages of simple operation, low cost, no need for complicated instruments, high sensitivity and good specificity.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(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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