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Detection probe for hepatitis B virus adefovir dipivoxil medicament-resistant variant strains and application method thereof

A technology for detecting probes and hepatitis B virus, applied in the medical field, can solve the problems of limited promotion and application, high price, low detection throughput, etc.

Inactive Publication Date: 2011-03-23
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sequencing analysis method requires an expensive sequencer, and the detection process takes a long time, and only when the HBV variant strains account for at least 30% (usually more than 50%) in the virus group, the variant strains can be detected, which is used for the detection of variant strains. Early detection has great limitations; the method based on PCR-RFLP may only detect one type of variation at a time; while the INNO-LiPA probe detection method has high sensitivity, but the detection cost is high, and the operation is complicated and time-consuming , the detection throughput is low, and at the same time, the price is expensive, and the popularization and application are limited

Method used

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  • Detection probe for hepatitis B virus adefovir dipivoxil medicament-resistant variant strains and application method thereof
  • Detection probe for hepatitis B virus adefovir dipivoxil medicament-resistant variant strains and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The determination of the detection probe of embodiment 1 hepatitis B virus adefovir drug-resistant variant strain

[0052] The present invention uses bioinformatics to determine the polymorphism of the adefovir resistance site of hepatitis B virus, as shown in Table 1 specifically.

[0053] Table 1.181 and 236 polymorphism analysis

[0054] Drug Mutation type Nucleotide before mutation Nucleotide after mutation

[0055] ADV rtA181T GCT ACT

[0056] rtA181V GCT GTT

[0057] rtA181S GCT TCC

[0058] rtN236T AAC ACC

[0059] Then design detection probes according to the adefovir resistance site of hepatitis B virus obtained in Table 1, after testing, all of SEQ ID NO 6-19 in Table 2 can be used as detection probes.

[0060] Table 2. Probe sequences

[0061]

Embodiment 2

[0062] Example 2 Detection of Hepatitis B Virus Adefovir-resistant Mutant Strains

[0063] 1. Sample pretreatment: HBV DNA, as a template for amplification;

[0064] 2. PCR amplification and biotin labeling of target fragments: clinical samples with a high viral load use HBV DNA as a template, and directly amplify with the inner primer (the 5' end of the downstream primer is biotin-labeled), including rt180 to 250 The sequence between amino acids, such as clinical samples with a high viral load, requires nested PCR, that is, use primers RTS1 and RTAS1 to amplify the RT region, and then use the inner primers of each site to perform the second step on the main variation region of the corresponding gene. Two rounds of PCR amplification (primers are listed in Table 3); PCR amplification conditions were: pre-denaturation at 94°C for 5 min; 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and extension at 72°C for 7 min after the last cycle. The reaction system was 25u...

Embodiment 3

[0084] Example 3 Serum Sample Detection

[0085] Collect blood samples from patients with hepatitis B, use phenol chloroform to extract serum HBV DNA, and use it as an amplification template;

[0086] According to the method of Example 2, detection is carried out, and the results are shown in Table 5.

[0087] Table 5 clinical serum sample detection result of the present invention method

[0088] 181A 181A-1 181S-1 181S-2 181T 181T-1 181V 181V-1 236N 236N-2 236N-3 236T 236T-2 236T-3 CON

[0089] Book

[0090] 1 59 18 1 10 7 1 4 5 118 7 39 9 5 29 100

[0091] 2 20 15 6 6 7 4 3 8 99 5 7 36 4 7 100

[0092] 3 1 13 2 7 10 42 3 2 18 5 6 118 4 7 100

[0093] 4 1 5 2 8 12 45 4 3 8 6 18 4 4 49 100

[0094] 5 29 4 1 8 53 9 5 1 4 38 6 4 56 5 100

[0095] 6 30 12 13 8 15 4 6 9 50 6 20 68 4 12 100

[0096] 7 5 3 3 22 10 2 8 3 12 17 86 11 11 25 100

[0097] 8 56 11 5 11 6 1 4 11 85 8 65 47 5 7 100

[0098] 9 5 57 1 5 3 1 2 1 94 4 4 3 3 3 100

[0099] 10 1 7 33 7 3 1 4 19 97 6 6 ...

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Abstract

The invention belongs to the field of medicament, and relates to a detection probe for hepatitis B virus adefovir dipivoxil medicament-resistant variant strains and an application method thereof. The detection probe for the hepatitis B virus adefovir dipivoxil medicament-resistant variant strains is one or more of SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18 and SEQ ID No.19. By combining liquid phase chip technology, the detection probe can quickly and accurately determine the hepatitis B virus lamivudine medicament resistance of a sample, has high specificity, good sensitivity and high detection flux, and greatly saves the cost for determining the category of the medicament-resistant variant strains.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to a detection probe for adefovir drug-resistant mutant strain of hepatitis B virus and clinical monitoring of drug resistance of chronic hepatitis B patients after adefovir antiviral treatment. Background technique [0002] Adefovir dipivoxil (ADV) is a ring-opening purine nucleotide analogue, which not only has a good curative effect on patients with chronic hepatitis B, but also has a good inhibitory effect on adefovir-resistant HBV , has been widely used in the treatment of chronic hepatitis B patients (including adefovir-resistant patients). Although the incidence of HBV resistance to adefovir is lower than that of lamivudine, there is still a high resistance rate after long-term use. For newly diagnosed patients, the resistance rates of ADV treatment for 5 years are 0, 2 % to 3%, 5.9% to 11%, 18%, 28%; for patients who switched to ADV monotherapy after LAM treatment failure, the drug re...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 张继明刘红艳李义良毛日成尹永喜夏佳慧范丽丽李新艳
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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