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Hepatitis B virus Adefovir dipivoxil drug-resistance nucleic acid quantitative detection reagent kit, detection method, primers and probes thereof

A technology of hepatitis B virus and adefovir dipivoxil, applied in the field of biological detection, can solve the problem of high cost

Active Publication Date: 2012-01-04
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since pyrosequencing technology can only accurately detect bases of about 40 bp at a time, it cannot replace conventional sequencing technology, and the cost of this technology is higher than that of Sanger sequencing method, and corresponding specific primers should be designed for different mutation sites It can only be detected and other shortcomings; there is no report on the detection of ADV drug resistance by pyrosequencing
[0014] In the prior art, there is no fluorescent quantitative PCR detection nucleic acid kit for hepatitis B virus adefovir dipivoxil resistance

Method used

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  • Hepatitis B virus Adefovir dipivoxil drug-resistance nucleic acid quantitative detection reagent kit, detection method, primers and probes thereof
  • Hepatitis B virus Adefovir dipivoxil drug-resistance nucleic acid quantitative detection reagent kit, detection method, primers and probes thereof
  • Hepatitis B virus Adefovir dipivoxil drug-resistance nucleic acid quantitative detection reagent kit, detection method, primers and probes thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] The method for detecting hepatitis B virus adefovir dipivoxil drug-resistant nucleic acid on ABI 7300 fluorescent quantitative PCR instrument with kit of the present invention:

[0146] (1) Collect samples: collect human serum samples;

[0147] 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.

[0148] 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.

[0149] (2) Sample treatment: Take 100 μL of the serum to be tested, add an equal amount of 15% solution A, mix well, centrif...

Embodiment 2

[0166] The kit of the present invention is used to quantitatively detect the adefovir dipivoxil drug-resistant nucleic acid of hepatitis B virus in an unknown sample according to the method of Example 1. The 60 unknown samples came from the blood samples of patients infected with hepatitis B virus in XX hospital.

[0167] The adefovir dipivoxil resistance site rt181 site 181A / 181V / 181T of the embodiment 2 of the present invention detects the fluorescent quantitative PCR experiment result and standard curve figure of the sample respectively Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E and Figure 5F Shown, wherein, A, B represent the amplification curve and standard curve of rt181A (wild strain) respectively; C, D represent the amplification curve and standard curve of rt181V (mutant strain) respectively; E, F represent rt181T (mutant strain) respectively strain) amplification curve and standard curve; as can be seen from the figure, the curve is smooth S...

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Abstract

The invention discloses a hepatitis B virus Adefovir dipivoxil drug-resistant nucleic acid quantitative detection reagent kit, a detection method, primers and probes thereof. The reagent kit contains fluorescence quantitative PCR (Polymerase Chain Reaction) reaction liquid, including DEPC (Diethyl Pyrocarbonate) treated water, DNA (Deoxyribose Nucleic Acid) polymerases with the 5'->3' exonucleolytic activity, dNTPs (Deoxyribonucleotide Triphosphates), 10*fluorescence quantitative PCR Buffer, solution containing Mg2+ions, rt181 site forward primers, rt181 site reverse primers, rt181A site probes, rt181V site probes, rt181T site probes as well as rt236 site forward primers, rt236 site reverse primers, rt236N site probes and rt236 site probes peculiar to a rtN236T detection reagent kit; and DNA extracting solution, negative controls, working standards, positive controls and critical positive controls are also contained in the reagent kit. According to the detection method, the fluorescence quantitative PCR (Polymerase Chain Reaction) technology is adopted for quantitatively detecting hepatitis B virus Adefovir dipivoxil drug-resistant strains; according to the fluorescence quantitative PCR (Polymerase Chain Reaction) method, the step of PCR (Polymerase Chain Reaction) extension is avoided; and the probes can mark a variety of fluorescent marks; and meanwhile, the reagent kit has the advantages of specificity, sensitivity, quickness and convenience in operation.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a quantitative detection kit for hepatitis B virus adefovir dipivoxil drug-resistant nucleic acid, a detection method, primers and probes thereof. Background technique [0002] Hepatitis B virus can cause chronic hepatitis, liver cirrhosis and primary liver cancer in the human body. Antiviral therapy is the main measure for the treatment of hepatitis B virus; nucleoside analogs are the most common drug for the treatment of hepatitis B virus, adefovir dipivoxil As a new nucleoside anti-HBV drug, it entered clinical treatment in my country in 2005. Adefovir dipivoxil is also a good remedy for lamivudine treatment ineffectiveness, which can inhibit the hepatitis B virus polymerase or compete to participate in the replication of hepatitis B virus, thereby stopping the elongation of the DNA chain of hepatitis B virus; however, due to the existence of cccDNA, The cure of h...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 唐景峰王业富刘绪
Owner WUHAN BIOTECH GENE ENG
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