Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus

A hepatitis B virus and nucleotide technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as high technical difficulty, PCR products that cannot be too long, and false negative results

Inactive Publication Date: 2012-08-08
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although its detection sensitivity is high, since each detection site needs to establish an independent enzyme cleavage reaction system, the operation is cumbersome, and not every drug resistance site can find the corresponding enzyme cleavage site, which is technically difficult
[0008] (3) Single-strand conformational polymorphism analysis (SSCP): When monitoring the emergence and dynamic changes of drug-resistant strains of viruses, mutations of single bases can be detected, but the amplified PCR products should not be too long, preferably in About 150bp, and if the point mutation occurs at both ends of the amplified fragment, the spatial conformation caused by the point mutation is very small, resulting in almost the same electrophoretic mobility, and false negative results are prone to occur
[0009] (4) Real-time fluorescent PCR: detect HBV mutants with different drug-resistant sites by different melting temperatures, but it is difficult to analyze multiple sites or adjacent sites
[0010] (5) Gene chip technology: high-throughput, multi-site detection of drug-resistant mutations of HBV is possible, but the detection of amplified products requires special instruments, well-trained technicians and multi-step washing and incubation steps, and the process is complicated and time-consuming
These detection methods are fast and accurate, but require expensive instruments, high work intensity, high technical requirements for operators, and are not suitable for mass screening, and are only used for laboratory analysis

Method used

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  • Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus
  • Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus
  • Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1 extracts HBV DNA gene from serum or plasma to be tested

[0082] Take 200ul virus serum + 200ul 2x lysis buffer (0.02mol / L Tris-HCl, 0.01mol / L EDTA, 1% SDS) + 10ul proteinase K (20mg / ml), digest at 40°C for 1.5 hours. Use phenol Extract with phenol: chloroform (1:1) and chloroform once each, mix well, 12000g x 3min, recover the aqueous phase, that is, the upper layer. Add 1 / 10 volume of 3mol / L sodium acetate and 2 times the volume of pre-cooled absolute ethanol to precipitate DNA, 12000g x 10 minutes, recover DNA, wash DNA once with 75% ethanol, discard supernatant, after drying, add Dissolve DNA in 20 μl of pure water, take 5 μl as template for PCR reaction, and store the rest at -20°C.

Embodiment 2

[0083] Example 2 Amplification of target DNA drug-resistant regions using digoxin-labeled oligonucleotide primers (nested PCR)

[0084] (1) External amplification of target DNA

[0085] Internal amplification primers:

[0086] HBV 570F 5'-TGTTGCTGTACAAAACCT-3' (SEQ NO 1)

[0087] HBV 1180R 5'-TCAGCAAACACTTGGCA-3' (SEQ NO 2)

[0088] 1. Test tube number (N): N = number of sample numbers + 1 negative control

[0089] 2. PCR reaction system (TaKaRa)

[0090]

[0091] Note: The whole process was operated on ice, and the negative control was supplemented by adding 5 μl of distilled water. After adding the template, mix well and centrifuge, then place it on the PCR reaction instrument.

[0092] 3. PCR cycle system:

[0093]

[0094] 4. Electrophoresis externally amplified PCR product: After completing the PCR cycle, immediately take 6 μL of PCR product and add corresponding DNA LoadingBuffer, electrophoresis on 1% agarose gel, and detect the presence or absence of bands, ...

Embodiment 3

[0106] The preparation of embodiment 3 hybridized nylon film strips

[0107] Dilute each artificially synthesized probe to a final concentration of 10 pmol / μl, and fix it on the corresponding position of the membrane strip. The preparation of the membrane strip is as follows:

[0108] (1) Add 27 specific probes, two samples at a time to the No. 1 pump and No. 2 pump of the automatic film spraying machine. After spraying, clean the two pipes with distilled water, and then replace the other two probes. Needle spray film.

[0109] (2) Immobilizing the probe: After the probe is fixed, after the probe is naturally dried at room temperature, it is subjected to ultraviolet cross-linking, and then baked at 80° C. for 30 minutes. The oligonucleotide probe is added with a polynucleotide tail chain, and 20 bases T are added to both the 5' and 3' ends of the probe.

[0110] (3) Cutting: After fixing, cut the film into 3mm×7cm film strips with a strip cutter along the marking line.

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Abstract

The invention provides a method and a reagent kit for simultaneously detecting the resistance sites of the three nucleotide analogues of a hepatitis B virus (HBV). Based on the genotype sequence of the HBV, four nested amplification primers and twenty-seven wild resistance-detection oligonucleotides probes aiming at ten resistance sites are designed in an HBV polymerase area, digoxin-labeled oligonucleotides universal primers are used for conducting nested PRC reaction to amplify a target DNA segment, a target DNA amplification product to be labeled is used for hybridizing with specific oligonucleotide probes on matrix, and the existence of the resistance of the HBV to the three nucleotide analogues is judged through enzymatic color development reaction link-coupled by hybridization conjugates. The method improves the accuracy, the reliability and the sensitivity and can simultaneously detect the ten resistance sites of the three nucleotide analogues, thereby realizing the high-throughput, multi-site, economic and rapid detection and fitting to clinical needs in a better way. The method is of great significance to the realization of early detection and the proper guide of clinicalpersonalized medication.

Description

technical field [0001] The invention relates to a method for detecting drug resistance of hepatitis B virus, in particular to a method for detecting drug resistance of hepatitis B virus to three types of nucleotide analogues by reverse dot hybridization technology, and also relates to a kit for clinical testing. Background technique [0002] Hepatitis B virus infection is widespread worldwide, especially in my country, which accounts for 1 / 3 of the world's infected people. 5%-10% of chronically infected patients can lead to liver cancer (HCC), 30% progress to liver cirrhosis, and 23% of patients with liver cirrhosis will develop liver failure within 5 years. According to statistics, the annual medical and health care expenses for HBV-infected patients in my country are as high as 100 billion RMB. Therefore, HBV infection seriously endangers people's health and causes serious losses to the national economy. [0003] Anti-HBV drug therapy has been recognized internationally ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 黄爱龙张文露胡源赖国旗刘彦辰赵丽
Owner CHONGQING MEDICAL UNIVERSITY
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