A kind of droplet digital PCR method and kit for clinical detection of hbv cccDNA
A kit and reagent technology, applied in the field of droplet digital PCR method and kit for clinical detection of HBV cccDNA, can solve the problems of low sensitivity, low detection sensitivity, limited clinical application, etc., and achieve improved sensitivity, good specificity, Source-rich effects
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Embodiment 1
[0047] Example 1 ddPCR detection cccDNA positive quality control
[0048] (1) PSAD enzyme digestion: take the positive quality control in the kit, use ddH 2 O after 10-fold serial dilution, PSAD enzyme digestion, the reaction system is 1 μL of PSAD enzyme, 1 μL of 10×PSAD buffer, 2 μL of ATP solution, 5 μL of DNA sample and ddH 2 O 1 μL; the reaction conditions were 37°C for 60 min and 70°C for 30 min.
[0049] (2) Preparation of ddPCR reaction system: DNA samples digested by PSAD were prepared according to the system shown in Table 1 below for droplet generation.
[0050] Table 1 ddPCR reaction system
[0051]
[0052] (3) Preparation of microdroplets: transfer the prepared ddPCR reaction solution into the DG8 microdroplet generator card, add 70 μL of microdroplet generator oil (Bio-Rad, USA) to each well, and put the microdroplet generator card into the microdroplet generator In the container, prepare microdroplets.
[0053] (4) ddPCR amplification: Add the prepar...
Embodiment 2
[0058] Example 2 ddPCR specific detection of cccDNA
[0059] (1) Take two samples of HepG2.2.15 cell DNA and serum DNA samples of hepatitis patients.
[0060] (2) Specificity verification of cccDNA detection: Take 5 μL of the DNA sample to be tested, digest it with PSAD enzyme, and use it for cccDNA and rcDNA detection; another 5 μL of the DNA sample to be tested is directly amplified to detect cccDNA and rcDNA. The PSAD enzyme digestion reaction system is 1 μL of PSAD enzyme, 1 μL of 10×PSAD buffer, 2 μL of ATP solution, 5 μL of DNA sample and ddH 2 O 1 μL; the reaction conditions were 37°C for 60 min and 70°C for 30 min.
[0061] The rcDNA detection primer pair is: rcDNA-F, 5'-CACTCTATGGAAGGCGGGTA-3'; rcDNA-R, 5'-TGCTCCAGCTCCTACCTTGT-3'.
[0062] The cccDNA and rcDNA detection reaction system is shown in Table 3 below:
[0063] Table 3 cccDNA and rcDNA detection reaction system
[0064]
[0065] The PCR amplification reaction conditions are shown in Table 4:
[006...
Embodiment 3
[0070] Example 3 Detection of cccDNA content in a single HepG2.2.15 cell by ddPCR
[0071] (1) Cell collection: when the cell coverage in the culture flask reaches 70%-80%, the cells are digested and collected. Discard the culture medium, add 1mL PBS to wash the cells twice, add 1mL of 0.25% trypsin solution, digest at room temperature for 3 minutes, discard the trypsin solution, add 1mL of medium containing serum to stop the digestion, gently pipette, and collect the cells to 1.5mL EP The tube was centrifuged at 1000rpm for 5min. Discard the supernatant, resuspend the cells with PBS solution, and adjust the concentration to 10 4 cells / mL.
[0072] (2) Obtain and lyse a single cell: pipette 10 μL of cell suspension onto the stage of TransferMan NK2, use the CellTram microinjector and VacuTip suction needle, pick 1, 2, 4, and 8 cells into different PCR tubes, and set 4 parallel tubes, 2 tubes for cccDNA detection, 2 tubes for β-actin detection. Subsequently, the PCR tub...
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