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Probe for detecting lamivudine drug-resistant variant of hepatitis B virus and application method thereof

A technology of lamivudine and detection probes, which is applied in the medical field, can solve the problems of long detection process, limited popularization and application, complex and time-consuming operation, etc., and achieve the effects of saving treatment costs, good sensitivity, and large detection throughput

Inactive Publication Date: 2011-03-23
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • 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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  • Probe for detecting lamivudine drug-resistant variant of hepatitis B virus and application method thereof
  • Probe for detecting lamivudine drug-resistant variant of hepatitis B virus and application method thereof
  • Probe for detecting lamivudine drug-resistant variant of hepatitis B virus and application method thereof

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Experimental program
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Effect test

Embodiment 1

[0055] The determination of the detection probe of embodiment 1 hepatitis B virus lamivudine drug-resistant variant

[0056] The present invention uses bioinformatics to determine the lamivudine resistance sites of hepatitis B virus as shown in Table 1, the nucleotide sequences at the 180th, 204th and nearby RT regions of HBV.

[0057] Table 1 The nucleotide sequences of the 180th, 204th and nearby RT regions of the constructed HBV full gene plasmid

[0058]

[0059] Note: The italic letters represent the codons at rt180 and rt204 respectively

[0060] Then according to the obtained hepatitis B virus lamivudine drug-resistant site design detection probe in Table 1, after testing,

[0061] SEQ ID NO 12-19 of Table 2 can all be used as detection probes.

[0062] Table 2. Probe sequences

[0063]

Embodiment 2

[0064] Example 2 Detection of Hepatitis B Virus Lamivudine-resistant Mutants

[0065] 1. Sample pretreatment: HBV DNA, as a template for amplification;

[0066] 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 (for primers, see the "Primer Sequence" table in the Summary of the Invention); PCR amplification conditions are: 94°C pre-denaturation for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 45s, cycle 35 times; the last cycle ends Then extend at 72°C for...

Embodiment 3

[0073] Example 3 Serum Sample Detection

[0074] Blood samples were collected from patients, and serum HBV DNA was extracted with phenol-chloroform as an amplification template; detection was performed according to the method in Example 2, and the results are shown in Table 4.

[0075] Table 4 MASA detection of serum samples from patients with lamivudine-related HBV resistance

[0076]

[0077] Note: Fluorescence value = target gene fluorescence value / internal reference fluorescence value × 100, the negative and positive hybridization signal values ​​of serum samples are represented by the median, Z value = -4.100, -4.588, -4.520, P<0.05, The difference was statistically significant.

[0078] The above results show that the detection results are consistent with the actual.

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Abstract

The invention belongs to the field of medical science and relates to a probe for detecting a lamivudine drug-resistant variant of a hepatitis B virus and an application method thereof. The probe for detecting the lamivudine drug-resistant variant of the hepatitis B virus is one or more than one of SEQ ID NO12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18 or SEQ ID NO 19. By combining the detection probe with a Luminex technology, the lamivudine drug resistance of the hepatitis B virus can be rapidly and accurately determined, advantages of high specificity, good sensitivity and large detection flux can be achieved, and the cost required by determining the variety of the drug-resistant variant is greatly saved.

Description

technical field [0001] The invention belongs to the medical field, and relates to a detection probe for a lamivudine drug-resistant mutant strain of hepatitis B virus and clinical monitoring of drug resistance of chronic hepatitis B patients after lamivudine antiviral treatment. Background technique [0002] The clinical application of nucleoside analog anti-hepatitis B virus (HBV) drugs represented by lamivudine (LAM) is a milestone in the history of chronic hepatitis B treatment. So far, more than 1 million chronic hepatitis B patients have received lamivudine treatment, which has made great progress in the treatment of chronic hepatitis B. While the wide application of anti-HBV drugs has brought good news to patients with chronic hepatitis B (CHB), it has also brought serious drug resistance problems. For example, the resistance rates of lamivudine treatment for 1 to 4 years can be as high as 14%, 38%, 49% and 66%, respectively. The main cause of HBV resistance to lamiv...

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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