HBV YMDD mutation, precore region mutation/BCP region mutation and genotyping integrated-detection DNA microarray chip
A technology of microarray chip and mutation detection, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve complicated, costly and time-consuming problems
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Embodiment 1
[0036] Example 1: Design of DNA microarray chip PCR primers and probes for integrated detection of HBV YMDD mutation, pre-C region / BCP mutation and genotyping
[0037] (1) HBV typing: design one upstream primer, two downstream primers, and the downstream primer CY3 fluorescent label, adopt asymmetric PCR amplification system; design 15 specific probes for HBV A, B, C, D typing, These four types are covered.
[0038] (2) HBVYMDD mutation: In order to specifically and efficiently amplify the target gene in the nucleic acid sample to be tested, the whole genome DNA sequence of hepatitis B virus A to H was retrieved from the Gene Bank, targeting the reverse transcriptase gene of hepatitis B virus For sites that are prone to mutation under the selection pressure of nucleotide analogue drugs, use primerpremier5.0 and oligo6.0 software to design two forward primers, one reverse primer, and reverse primer CY3 fluorescent label, and use asymmetric PCR reaction system to amplify the hy...
Embodiment 2
[0040] Example 2: Preparation of DNA microarray chip for integrated detection of HBV YMDD mutation, pre-C region / BCP region mutation and genotyping
[0041] Using a gene chip automatic spotting instrument, 42 detection probes and 6 control probes (the control probes are positive and negative control probes designed for the three detection target conservative regions respectively) were spotted on specific areas of the slide. area to make a DNA microarray.
[0042] Spotting probe solution: dilute the probe into 5×SSC, 0.05% SDS solution, the final concentration is 30 μM;
[0043] Spotting matrix: (see attached figure 1 ) spot the probes on the glass slide, and repeat 2 points of each probe horizontally in the hybridization area, 12 points in each row, 8 rows in total, 96 points; the hybridization area is divided into 10 areas, and 8 samples can be detected at the same time (9,10 hybridization area is the control area).
Embodiment 3
[0044] Embodiment 3: Using the product of the present invention to detect 8 clinical samples
[0045] Take 8 samples and 2 controls, extract viral DNA, first perform DNA sequencing, determine HBV typing, drug resistance and pre-C region / BCP region mutation results, and then use the product of the present invention to detect and verify.
[0046] The DNA microarray chip of the present invention is used to perform one-time detection on the above 8 samples. Take 10 tubes of multiplex PCR reaction solution, add 10 tubes of extracted DNA respectively, 5ul / tube, the total reaction system is 50ul, the PCR cycle parameters are: 94°C 5'; 94°C 30", 55°C 30", 72°C 30", 45 cycles; 72°C 5'.
[0047] Take a microarray chip, add 2ul of 10 tubes of PCR products to the 10 wells of the chip, then add 8μl of hybridization solution (5×SSC) to each well, put the chip into the tank of the automatic hybridization washing machine, and set the hybridization The temperature was 50°C, and the hybridiza...
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