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Hepatitis B virus lamivudine resistant RNA quantitative detection primers and probes

A technology of hepatitis B virus and lamivudine, which is applied to the determination/testing of microorganisms, microorganisms, methods based on microorganisms, etc., can solve problems such as detection failures and false positives, and achieve reliable results, easy operation, and automation high effect

Active Publication Date: 2015-02-04
WUHAN BIOTECH GENE ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the specificity of PCR amplification is poor, it will lead to false positives, so that the detection will fail
[0016] In the prior art, there is no fluorescent quantitative PCR detection kit for hepatitis B virus lamivudine drug-resistant nucleic acid

Method used

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  • Hepatitis B virus lamivudine resistant RNA quantitative detection primers and probes
  • Hepatitis B virus lamivudine resistant RNA quantitative detection primers and probes
  • Hepatitis B virus lamivudine resistant RNA quantitative detection primers and probes

Examples

Experimental program
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Effect test

Embodiment 1

[0090] Method for detecting hepatitis B virus lamivudine drug-resistant nucleic acid on ABI7300 fluorescent quantitative PCR instrument with kit of the present invention

[0091] (1) Collect samples: collect human serum samples;

[0092] a. Use a sterile syringe to extract 1ml of venous blood from the subject, inject it into a sterile 1.5ml centrifuge tube, let it stand at room temperature for 2 hours, transfer it to 4°C for 1 hour, centrifuge at 8000rpm for 5 minutes, and draw 200μL of supernatant (note: Do not inhale red blood cells), transfer to another sterile 1.5ml centrifuge tube, which is the serum sample.

[0093] b. Specimen storage and transportation: If the specimen is not tested immediately, it should be stored at -20°C and avoid repeated freezing and thawing. Long-distance transportation of specimens should use 0 ℃ curling.

[0094] (2) Sample treatment: Take 100 μL of the serum to be tested, add an equal amount of 15% solution A, mix well, centrifuge at 13000 r...

Embodiment 2

[0112] According to the method of Example 1, the kit of the present invention is used to quantitatively detect the hepatitis B virus lamivudine drug-resistant nucleic acid of the unknown sample. The 60 unknown samples came from the hepatitis B virus samples to be tested for drug resistance in XX hospital patients.

[0113] The results of the YMDD site probe in Example 2 of the present invention are respectively to the results of the sample fluorescent quantitative PCR test as shown in Figure 5, wherein, A, B: respectively represent the amplification curve and standard curve of rt204M (wild strain); C, D : Represent the amplification curve and standard curve of rt204I (mutant strain) respectively; E, F: Represent the amplification curve and standard curve of rt204V (mutant strain) respectively; As can be seen from the figure, the curve is smooth S-shaped, and the amplification effect better. The results of the rt180 site probes on the sample fluorescent quantitative PCR test a...

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Abstract

The invention discloses a hepatitis B virus lamivudine resistant RNA quantitative detection kit, a detection method, primers and probes. The kit comprises fluorescent quantitative polymerase chain reaction (PCR) solution which contains diethyl pyrocarbonate (DEPC) treatment water, DNA polymerase with 5'-3' exonucleolytic activity, dNTPs, 10X fluorescent quantitative PCR Buffer, solution containing Mg<2+>, rt204 locus forward primer, a rt204 locus backward primer, a rt204M locus probe, a rt204I locus probe, a rt204V locus probe, a rt180 locus forward primer, a rt180 locus backward primer, a rt180L locus probe and a rt180M locus probe. The kit also comprises DNA extraction solution, a negative control, a working standard, a positive control and a critical positive control. In the invention, a real-time fluorescent quantitative PCR technique is adopted to quantitatively detect a lamivudine resistant hepatitis B virus strain, the fluorescent quantitative PCR method is changed into a '2-step method', and the step of PCR extension is avoided; and the probes can mark various fluorescent marks and have the advantages of specificity, sensitiveness, quickness and simple and convenient operation.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer and a probe for the quantitative detection of hepatitis B virus lamivudine drug-resistant nucleic acid. Background technique [0002] Hepatitis B virus (HBV) is a highly dangerous virus. Hepatitis B virus infection is widespread in the world. Hepatitis B virus infection can not only lead to acute and chronic viral hepatitis, but also develop into liver cirrhosis and hepatocellular carcinoma. Caused about 1 million deaths and seriously affected human health. After the hepatitis B virus adsorbs and enters the liver cells, it takes off its capsid, and under the action of DNA polymerase, the negative strand DNA is used as a template to repair the gap of the positive strand, thereby forming a covalently closed circular double-stranded DNA that enters the nucleus for transcription. During the replication process of hepatitis B virus, because the reverse transcrip...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 唐景峰王业富刘绪
Owner WUHAN BIOTECH GENE ENG
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