Detection kit for nucleoside analogue drug-resistant correlated mutation of hepatitis B viruses
A technology of hepatitis B virus and nucleoside analogs, which is applied in the field of hepatitis B virus nucleoside analog drug resistance-related mutation detection kits, can solve the problems of cumbersome operation, high cost, and unfavorable development of primary hospitals, and achieve operational The effect of simplicity, low price, good application prospects and generalizability
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Embodiment 1
[0093] Embodiment 1: the extraction of serum DNA
[0094] Aseptically collect about 1ml of venous blood from the hepatitis B patient to be tested, let it stand at room temperature and 4°C for 2 hours and 1 hour respectively, then centrifuge (8,000rpm, 5 minutes) to separate and collect 200μl of serum sample. Then take 40 μl of serum sample, add an equal amount of DNA extraction solution to it, mix thoroughly and place in a boiling water bath for 10 minutes. In order to ensure that the virus particles contained in the serum can be fully lysed, the mixture was further left to stand at 4° C. for 10 hours. It was then centrifuged (10,000 rpm, 5 minutes) and 2 μl of the supernatant was collected and used for further PCR reactions.
Embodiment 2
[0095] Example 2: Nested PCR amplification of a target DNA fragment comprising the region sequence of HBV DNA polymerase rt173-rt250.
[0096] Take several tubes of PCR reaction solution for one person, add 2 μl template (or negative and positive standard) directly to each tube, mix well and then centrifuge briefly (3 seconds). Then put each reaction tube into the PCR instrument, pretreatment at 50°C for 3 minutes, and amplify according to the following conditions: 93°C for 5 minutes, then 93°C for 30 seconds, 68°C for 1 minute, a total of 25 cycles, and then 93°C for 30 seconds. seconds, 55°C for 30 seconds, 72°C for 45 seconds, a total of 30 cycles, and finally 72°C for 7 minutes. Amplified products were detected by 2% agarose gel electrophoresis (see attached figure 2 ). The PCR amplification system used included: 1× qualitative PCR buffer, 0.2mM dNTPs, 6U Taq enzyme, 0.1pmol primer 1, primer 4 (SEQ ID NO:1,4) and 0.3pmol primer 2, primer 3 (SEQ ID NO: 2, 3).
Embodiment 3
[0097] Example 3: Reverse dot blot detection of samples of three genotypes by water bath hybridization
[0098] Before hybridization, adjust the temperature of the water bath shaker (or water bath) to the hybridization temperature, and take hybridization solution I (2×SSC-0.1% SDS) and hybridization solution II to preheat to 40-50°C for later use. Take several 15ml centrifuge tubes according to the number of samples to be tested, add 8-10ml hybridization solution I to each tube and preheat to 40-50°C. Then take 1000 μl hybridization solution I+2 μl solution I (1000:2) mixed solution as the binding solution and store it at 4°C for later use, and take the mixture of solution II: solution III: solution IV (1900:200:1) (1.9ml solution II+ 0.2ml of solution III + 1 μl of solution IV) is used as a chromogenic solution and stored away from light for later use.
[0099] First, add the preheated hybridization solution I into the tube until the membrane strip is covered, add the PCR pr...
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