Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV)
A genotype and kit technology, applied in the field of kits for detection of hepatitis C virus genotype and IL28 site polymorphism, can solve the problems of regional and ethnic differences in virus distribution, missed detection of the population, etc.
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Embodiment 1
[0039] The preparation of embodiment 1 sample target nucleic acid
[0040] Three cases of EDTA anticoagulated whole blood samples confirmed by sequencing were selected as HCV type 1b simultaneous IL28B polymorphic site rs12979860C, HCV type 2a simultaneous IL28B polymorphic site rs12979860C, and 3b type simultaneous IL28B polymorphic site rs12979860T, according to Roche company's extraction reagent High Pure Viral Nucleic Acid Kit instructions require the extraction of nucleic acids.
Embodiment 2
[0041] Embodiment 2 one-step RT-PCR reaction
[0042] The volume of RT-PCR reaction reagent per person is 50 μl, prepared according to Qiagen’s Onestep RT-PCR Kit (operated according to the instructions), including 1 μl 10mM HCV-RT, 1 μl 0.5mM HCV-F, 1 μl 5mM HCV-R, 1 μl 0.5 mM IL28B-F, 1 μl of 5 mM IL28B-R. Add 5 μl of the nucleic acid extracted from the specimen of Example 1 into the RT-PCR reaction reagent respectively, and perform amplification under one-step reaction conditions. 72°C for 45 seconds, run 45 cycles, and finally 72°C for 7min.
Embodiment 3
[0043] Embodiment 3 Two-step reverse transcription reaction
[0044] The volume of reverse transcription reagent per person is 25 μl, according to Ambion's RETROscript TM The kit (operated according to the instructions) was prepared, including 0.5 μl of 10mM HCV-RT, and 5 μl of the nucleic acid extracted from the specimen of Example 1 was added to the reverse transcription reagent respectively, and the reverse transcription reaction conditions were used for amplification. The specific conditions were 44°C 60 minutes, 10 minutes at 92°C.
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