Method for quickly detecting hepatitis B virus drug-resistant gene variation

A technology of hepatitis B virus and drug-resistant genes, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of mixed mutation detection, cumbersome experiments, and restrictions on wide application, so as to facilitate monitoring Efficacy, adjustment of treatment plan, effect of high amplification efficiency

Inactive Publication Date: 2006-02-22
SHANGHAI FUDAN YUEDA BIOTECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the different viral titers in patients with hepatitis B, the mutations that exist after lamivudine treatment are mixed mutations, that is, the symbiosis between YMDD, YIDD, and YVDD, and the ratio between them is different. High-copy strains will competitively cover up low-copy mutant strains. It is difficult to detect the exact mutation of the HBV gene by comparing the difference between the melting curve and the Tm value to determine the genetic variation, and it is powerless to detect mixed mutations.
Although the traditional sequencing technology is accurate, its wide application is limited due to shortcomings such as time-consuming, cumbersome experiments, and professional comparison of sequences.

Method used

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  • Method for quickly detecting hepatitis B virus drug-resistant gene variation
  • Method for quickly detecting hepatitis B virus drug-resistant gene variation
  • Method for quickly detecting hepatitis B virus drug-resistant gene variation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 preparation kit

[0045] The main components of the kit are PCR reaction mixture A (which also contains primers 1, 2 and fluorescent probe M), PCR reaction mixture B (which also contains primers 1, 2 and fluorescent probes I and V), Taq enzyme, HBV DNA Extraction Reagent, Negative Quality Control and ddH 2 O.

[0046] The primers and fluorescent probes are designed near the 552nd amino acid encoded in the P gene region of the hepatitis B virus, are only specific to the hepatitis B virus, and have no cross-reaction with other species. Primers were designed near the YMDD region of the HBV-encoded polymerase DNA sequence of EMBL, with a full length of 92 bps and a single-copy gene sequence. The fluorescent probe design site is located between the two primers.

[0047] The primer sequences are:

[0048] Primer 1: 5′GTA GGG CTT TCC CCC ACT G 3′19bps

[0049] Primer 2: 5′GGT AAA AAG GGA YTC AMG ATG 3′21bps The fluorescent probe sequences for YMDD, YIDD, and ...

Embodiment 2

[0075] Example 2 Using the Hepatitis B Virus Gene Drug Resistance Mutation Detection Kit for Mutation Detection

[0076] Take 50 μl of the serum sample to be tested, add 50 μl of HBV DNA extraction solution I, shake and mix for 15 seconds, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant. Add 50 μl of HBV DNA Extraction II solution, shake and mix well, and bathe in water at 100°C for 10 minutes. Centrifuge at 12000rpm for 2min, and take the supernatant for PCR amplification, or store it at -20°C for future use.

[0077] Negative quality control products were treated the same as above.

[0078] PCR amplification:

[0079] 2 μl of the HBV DNA-containing supernatant extracted by the above method was added to the PCR reaction mixture A and B, mixed evenly and placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad, iCycler), and the amplification reaction was carried out according to the following cycles: 94 Pre-denaturation at ℃ for 5 minutes...

Embodiment 3

[0087]Configure the PCR reaction solution as in Example 1, put the PCR tube into a fluorescence detector that can read the fluorescence, read the value, record the fluorescence value A0, transfer the PCR tube to an ordinary PCR instrument, and perform the amplification reaction according to the following cycle : 94°C pre-denaturation for 5 minutes → (94°C denaturation for 20 seconds → 60°C annealing for 40 seconds → 72°C extension for 30 seconds) × 40 cycles → 72°C extension for 2 minutes, then move the PCR product to a fluorescence detector to read the fluorescence Value Ax.

[0088] Analyzing the end-point fluorescence value, the following results can be judged:

[0089] 1. If the value of tube A (Ax-A0) of the PCR reaction mixture is higher than 10 times the standard deviation of the negative control, it is considered that the HBV DNA position 552 in the sample is wild-type YMDD;

[0090] 2. Tube B of the PCR reaction mixture has a fluorescent signal at the wavelength of F...

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Abstract

The invention, blonging to plant biotechnology field, relates a method of checking the virus of B hepatitis genovariation. The invention utilize the fluorescent light definite quantity PCR MGB Taqman probe technique, according to the YMDD nucleic acid sequence of B hepatitis virus, designing the genovariation of primer and probe checking HBV YMDD region. The invention adopts PCR reaction mixture A, B, Tap enzymes, HBV DNA extractant, and prepares diagnosis agent case after optimization and improvement with ddH20. The method can rapidly and exactly check the gene mutation situation of privileged site, quantitate the ratio of hybrid mutational virogene, quckly check the clinical hepatitis B drug therapia drug tolerance mutation. The method and agent case are not confined by medical equipment, so it is very easy to popularize.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for detecting hepatitis B virus related gene variation, in particular to a method for detecting hepatitis B virus lamivudine resistance related gene variation and a rapid diagnostic kit thereof. Background technique [0002] In the reports on the incidence of statutory Class A and B infectious diseases over the years, hepatitis B has always ranked first, seriously threatening human health, and it is estimated that 2 billion people around the world are infected. According to the latest figures released by the Ministry of Health in August 2003, by the end of 2002, there were 130 million hepatitis B virus carriers nationwide, and more than 20 million chronic hepatitis B patients, accounting for 1.57% of the total population. 50% to 75% of them have active virus replication and liver inflammation changes, and some chronic hepatitis can progress to liver cirrhosis, liver fa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 袁正宏王华耿海峰华兵邵醇
Owner SHANGHAI FUDAN YUEDA BIOTECH
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