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HBV phenotype drug resistance detection kit and preparation method thereof

A detection kit and drug resistance technology are applied in the preparation of HBV phenotype drug resistance detection kits and the field of phenotype drug resistance detection kits, achieving the effects of high sensitivity and easy operation.

Active Publication Date: 2013-09-25
盛世菁华(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first technical problem to be solved by the present invention is to provide a kind of phenotypic drug resistance detection kit for HBV and various existing NAs drugs

Method used

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  • HBV phenotype drug resistance detection kit and preparation method thereof
  • HBV phenotype drug resistance detection kit and preparation method thereof
  • HBV phenotype drug resistance detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1HB

[0044] The construction of embodiment 1HBV phenotype drug-resistant carrier

[0045] The construction steps of the HBV phenotypic drug resistance vector include producing a set of Gateway-based TM Recombinant expression vector system. The system includes a dual expression vector with secreted alkaline phosphatase and a recombinant entry vector that can replace HBV1.0 fragments. The main technical routes are as follows: Figure 8 shown.

[0046] Specific steps are as follows:

[0047] 1. Construction of pcDNA6.2-HBV1.1-sapI vector that can replace HBV1.0 fragment

[0048] The steps are specifically:

[0049] 1.1 First, amplify the multiple cloning site region of the pMD18T self-ligating vector, and the primers are as follows;

[0050] 6.2-salI-s: 5'-ACGCGTCGACCGAAAGGGGGATGTG-3' (shown in SEQ ID No.8);

[0051] 6.2-salI-as: 5'-ACGCTCGAGGAGCTCGGTACCCGG-3' (shown in SEQ ID No.9);

[0052] Use the pMD18T self-ligating carrier stored in our laboratory as a template for amplif...

Embodiment 2HB

[0071] Example 2 HBV drug resistance carrier system drug resistance detection application verification

[0072] 1. Substitution of the HBV1.0 fragment to be tested was introduced into the pcDNA6.2-HBV1.1-sapI vector constructed in Application Example 1 to form the entry clone pcDNA6.2-HBV1.1(p);

[0073] 1.1 Amplification of standard HBV C1 subtype HBV1.0 fragment

[0074] Primers used: HBV1.0-as: TAGTGGAAGCTTGACCATGGTGAGCAAGGGCGAG (shown in SEQ ID No.16);

[0075] HBV1.0-s: GCTTGGGATCCAATTCTTACTTGTACAGCTCG (shown in SEQ ID No.17);

[0076] Preserve the HBV C2 subtype plasmid template in the laboratory, reaction conditions: 94 degrees for 2 minutes; 94 degrees for 15 seconds, 55 degrees for 30 seconds, 72 degrees for 3 minutes and 20 seconds for 30 cycles of amplification; 72 degrees for 10 minutes;

[0077] 1.2 The amplified product and the pcDNA6.2-HBV1.1-sapI vector were digested with sapI and SalI and ligated to form pcDNA6.2-HBV1.1(p).

[0078] 2. Recombination: clonin...

Embodiment 3

[0085] Embodiment 3 Description about the kit

[0086] In the kit provided by the present invention, other components or structures such as primers, probes, chromogenic reagents and the like are also included. The specific instructions are as follows:

[0087] pcDNA6.2-HBV1.1-sapI vector 1 μg

[0088] pcDNA5AP-attR vector 1 μg

[0089] Primer HBV1.0-as TAGTGGAAGCTTGACCATGGTGAGCAAGGGCGAG (shown in SEQ ID No.16)

[0090] Primer HBV1.0-s GCTTGGGATCCAATTCTTACTTGTACAGCTCG (shown in SEQ ID No.17)

[0091] Other components of the kit, such as reagents for amplification PCR, etc., can be synthesized and purchased by themselves, and will not be described in detail here.

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Abstract

The invention discloses a HBV phenotype drug resistance detection kit, which comprises a recombinant entry vector pcDNA6.2-HBV1.1-sapI capable of replacing a HBV1.0 fragment and a double expression vector pcDNA5AP-attR having secreted alkaline phosphatase, wherein a base sequence of the recombinant entry vector pcDNA6.2-HBV1.1-sapI is represented by SEQ ID No.4, and a base sequence of the double expression vector pcDNA5AP-attR is represented by SEQ ID No.6. The drug resistance detection kit mainly comprises a set of an expression vector system based on GatewayTM recombination, wherein the recombination system has the following characteristics that: limitation of restriction endonuclease sites on clone can be avoided, a target fragment can be rapidly and correctly introduced into an expression vector, and a culture medium supernatant can be adopted to carry out chemiluminescence quantitative detection, such that sensitivity is high, quantification and calibration can be performed according to the standard alkaline phosphatase control, and operation is convenient and stable.

Description

technical field [0001] The invention relates to a drug resistance detection kit, in particular to a phenotypic drug resistance detection kit for HBV (hepatitis B virus) targeting various existing NAs drugs. At the same time, the invention also relates to the preparation method of the HBV phenotype drug resistance detection kit. Background technique [0002] Chronic hepatitis B (CHB) is an important medical and public health problem worldwide, currently affecting approximately 400 million people worldwide. Complications such as cirrhosis, decompensated liver disease, and hepatocellular carcinoma (HCC) may occur in up to 40% of CHB. [0003] Currently, drugs available for the treatment of chronic hepatitis B (CHB) include immunomodulators (such as interferon-α) and oral nucleoside / nucleotide analogs (NAs), including lamivudine, lamivudine, Telbivudine, Entecavir, and Tenofovir. Oral NAs have become the mainstay of chronic hepatitis B treatment due to their strong viral supp...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/85C12N15/66C12R1/93
Inventor 陈德喜乔录新魏飞力张玉林刘道洁刘秀红李庆丁渭徐树莹宋凤丽李宁
Owner 盛世菁华(北京)生物科技有限公司
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