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Primer probe and kit for detecting hepatitis B virus based on RAA fluorescence method

A hepatitis B virus, primer probe technology, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve the problems of high target gene, high false positive rate, cross-contamination, etc., to achieve high specificity, Good specificity and easy operation

Inactive Publication Date: 2018-12-21
JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, LAMP technology requires multiple pairs of primers and has high requirements for target genes, and the false positive rate is relatively high, which is prone to cross-contamination

Method used

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  • Primer probe and kit for detecting hepatitis B virus based on RAA fluorescence method
  • Primer probe and kit for detecting hepatitis B virus based on RAA fluorescence method
  • Primer probe and kit for detecting hepatitis B virus based on RAA fluorescence method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The present invention extracts the DNA of the hepatitis B virus sample for verification, and finds the corresponding full gene sequence (www.ncbi.nlm.nih.gov) in genebank according to the gene name hepatitis B virus (Hepatitis B virus, HBV), and uses DNAMAN The software performs homology analysis and blast sequence analysis, and screens out highly conserved sequences of hepatitis B virus (nt311-nt751, GenBank accession no. MH220971.1). According to the highly conserved sequence as the target gene for detection, the invention synthesizes the recombinant DNA plasmid of hepatitis B virus and designs primer probes.

[0040] According to the target gene, entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the recombinant DNA plasmid of hepatitis B virus, and the size of the plasmid is 440bp;

[0041] ①Primer probe design

[0042] According to the design principles of RAA technical primers and probes, through screening and evaluation, the final determination:

...

Embodiment 2

[0064] Sensitivity experiment

[0065] Upstream primers:

[0066] 5'-attcgcatgccccaacctcaatcactcacc-3';

[0067] Downstream primers:

[0068] 5'-aataccacatcatccatataactgaaagcc-3'.

[0069] The probe sequence is:

[0070] 5'-tatcatcttcctcttcatcctgctgctatgcctcatcttcttgtt ggttc-3'

[0071] The fluorescent reporter group uses FAM, and the fluorescent quencher group uses BHQ1;

[0072] The modified probe is:

[0073]5'-TATCATCTTCCTCTTCATCCTGCTGCTATGCCTCA(FAM-DT)(THF)(BHQ1-DT)TCTTGTTGGTTC-3';

[0074] The composition of the kit is shown in Table 1:

[0075] Table 1 Kit composition list

[0076]

[0077] The working standards of different concentrations prepared by the positive standard of hepatitis B virus recombinant DNA plasmid are:

[0078] Working standard (positive quality control) 1, containing 1.0×10 5 Copies / μL HBV recombinant DNA plasmid.

[0079] Working standard (positive quality control) 2, containing 1.0×10 4 Copies / μL HBV recombinant DNA plasmid.

[008...

Embodiment approach

[0084] 1 Preparation of reaction buffer

[0085] Draw 280 μL of reaction buffer from the reaction buffer tube in the kit and add it to the pre-prepared 1.5mL PE tube, then add 16 μL of the mixture of probe and primer (the concentration of the probe is 0.02mmol / L, and the concentration of the primer is 0.05mmol / L ), and mix thoroughly to obtain the mixed reaction buffer.

[0086] 2 RAA fluorescent basic reaction reagent redissolution

[0087] Prepare 6 RAA fluorescent basic reaction reagents, draw 49 μL of the reaction buffer mixed in step 1 and add them to the prepared 6 RAA fluorescent basic reaction reagent tubes, so that the lyophilized powder is fully dissolved and mixed to form an RAA reaction system .

[0088] 3. Sample addition reaction

[0089] Add 1 μL of negative quality control, 1 μL of working standard 5, 1 μL of working standard 4, 1 μL of working standard 3, 1 μL of working standard 2, and 1 μL of working standard into the above 6 prepared RAA fluorescent basi...

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Abstract

The invention provides a primer probe group for detecting hepatitis B virus based on RAA fluorescence method, and belongs to the technical field of molecular biological detection. The primer probe group comprises an upstream primer, a downstream primer and a probe, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown by SEQ ID NO.2, and the nucleotide sequence of the probe is shown by SEQ ID NO.3; the probe is a modified by a fluorescence reporter group and a fluorescence quenching group. By adopting the primer probe group provided by the invention, detection of the hepatitis B virus DNA can be finished within 15min, high temperature is not required for DNA unwinding, and the detection can be finished simply by performing isothermal amplification at 30-42 DEG C; moreover, the detection is fast and sensitive and has high specificity while avoiding false positive.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a primer probe group and a kit for detecting hepatitis B virus based on RAA fluorescence method. Background technique [0002] Hepatitis B virus (HBV) infection is still a global problem threatening people's health. At present, there are nearly 2 billion HBV-infected people in the world, among which more than 300 million are chronically infected. In the Asia-Pacific region, the chronic HBV infection rate exceeds 10%, and 25% to 40% of patients will die from liver cirrhosis with or without HCC. The World Health Organization has listed HBV infection as one of the top ten causes of death in the world. HBV infection can not only lead to acute and chronic viral hepatitis and severe hepatitis, but also closely related to the occurrence and development of liver cirrhosis and hepatocellular carcinoma. HBV infection causes 500,000 to 1,200,000 deaths wor...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/706C12Q1/6844C12Q2563/107C12Q2521/507
Inventor 申辛欣马学军蔡禹希郭利川汤赛君王智宏应清界
Owner JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD
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