Improved reverse dot hybridization method and application thereof
A technology of reverse dot hybridization and hybridization solution, which is applied in the application field of reverse dot hybridization method in products, can solve the problems of long operation time, many operation steps, and high price of automatic hybridization instrument, and achieve the goal of reducing steps and time Effect
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[0033] Example 1 Comparison between the traditional method and the method of the present invention in the detection of thalassemia
[0034] The beta thalassemia detection kit produced by Daan Gene Co., Ltd. of Sun Yat-sen University is used to compare the detection results of the method of the present invention and the kit method (ie, the traditional method).
[0035]
[0036]
[0037] The enzyme used in method A is horseradish peroxidase, and the enzyme used in method B is alkaline phosphatase. Three clinical samples were selected, namely CD41 / 42 heterozygotes, IVS2-654 heterozygotes, and β globin were normal. From the experimental results, method A, method B and kit method can all correctly detect the corresponding results, and the three are implemented There is no difference in effect (see attached figure 1 Numbers 1 to 3 are traditional methods, 4 to 6 are methods A of the present invention, and 7-9 are methods B) of the present invention. The method of the present invention ha...
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[0038] Example 2 Application of the method of the present invention in HCV genotyping detection
[0039] 1. The primer and probe sequences used in the kit are as follows:
[0040] Hepatitis C virus (HCV) nucleic acid (RNA) reverse transcription primer sequence is:
[0041] Reverse transcription (RT) primer: 5'-GCTCATGGTGCACGGTCTACGAGACCT-3' (SEQ ID NO: 1)
[0042] The upstream and downstream primer sequences used in the polymerase chain reaction amplification system are:
[0043] PCR upstream primer: 5'-TCTAGCCATGGCGTTAGTATGAGTGT-3' (SEQ ID NO: 2)
[0044] PCR downstream primer: 5'-CACTCGCAAGCACCCTATCAGGCAGT-3' (SEQ ID NO: 3)
[0045] The oligonucleotide probes used in the hybridization reaction are all labeled with an amino group at the 5'end, and the probe IC is labeled with an amino group at the 5'end and biotin at the 3'end. The sequences are:
[0046] Probe 1: 5'-CAATGCCTGGAGATTTGGGCG-3' (SEQ ID NO: 4)
[0047] Probe 1b-1: 5'-CCGCGAGACTGCTAGCCG-3' (SEQ ID NO: 5)
[0048] Probe 1b-2: 5'-...
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