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.
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[0044] Example 1 Construction of HBV phenotype drug-resistant vector
[0045] The construction steps of HBV phenotype resistant vector include generating 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-linking vector, 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-linked vector stored in our laboratory as a template for amplification. The amplified sequence is shown i...
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[0071] Example 2 HBV drug resistance vector system drug resistance detection application verification
[0072] 1. Replacement of the HBV1.0 fragment to be tested and introduced the pcDNA6.2-HBV1.1-sapI vector constructed in Example 1 to form the entry clone pcDNA6.2-HBV1.1(p);
[0073] 1.1 Amplify 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] Store 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 are digested with sapI and SalI and then connected to form pcDNA6.2-HBV1.1 (p).
[0078] 2. Recombination: gate clone pcDNA6.2-HBV1.1(p), pDONR221, us...
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[0085] Example 3 Description of the kit
[0086] The kit provided by the present invention also contains other components or structures such as primers, probes, color reagents, and the like. The specific instructions are as follows:
[0087] pcDNA6.2-HBV1.1-sapI vector 1μg
[0088] pcDNA5AP-attR vector 1μg
[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 amplification PCR reagents, etc. can be synthesized and purchased by yourself, and will not be described in detail here.
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