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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

Active Publication Date: 2011-06-22
DAAN GENE CO LTD
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  • Claims
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AI Technical Summary

Problems solved by technology

However, at present, the entire hybridization process of RDB technology requires many steps, and the operation time is relatively long, which has become a resistance to large-scale promotion and application.
Based on this demand, although corresponding automated hybridization instruments have appeared, the price of automated hybridization instruments is expensive, which is not acceptable to ordinary primary hospitals
Moreover, the numerous operating steps also pose a challenge to the stability of automated instrument operation
[0004] Therefore, it is necessary to improve the existing RDB technology to be more suitable for use in clinical testing. At present, there is no technical report related to the present invention both at home and abroad

Method used

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  • Improved reverse dot hybridization method and application thereof
  • Improved reverse dot hybridization method and application thereof
  • Improved reverse dot hybridization method and application thereof

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Effect test

Embodiment 1

[0033] Embodiment 1 Comparison between the traditional method and the method of the present invention in the detection of thalassemia

[0034] The β-thalassemia detection kit produced by Sun Yat-sen University Da'an Gene Co., Ltd. was used to compare the detection results of the method of the present invention and the method of the kit (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 heterozygote, IVS2-654 heterozygote, and normal β-globin. From the experimental results, method A, method B and the kit method can all detect the corresponding results correctly. There was no difference in effect (see appendix figure 1 , numbers 1-3 are traditional methods, 4-6 are method A of the present invention, and 7-9 are method B of the present invention, and the operation steps of the method of the present invent...

Embodiment 2

[0038] Embodiment 2 Application of the inventive method in HCV genotyping detection

[0039] 1. The primers 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 PCR 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 amino groups at the 5' end, where the 5' end of the probe IC is labeled with amino group, and the 3' end is labeled with biotin. 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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Abstract

The invention relates to an improved reverse dot hybridization method. By adopting the method, the steps in the conventional reverse dot hybridization method are optimized, so that the steps and time of reverse dot hybridization experiment operation are reduced without causing any difference on a detection result. The application of the method to a reverse dot hybridization product contributes to popularization of the method in the field of clinical detection.

Description

technical field [0001] The invention relates to an improved reverse dot hybridization method and the application of the improved reverse dot hybridization method to products. Background technique [0002] In 1989, reverse dot blot (RDB) technology was proposed by Saiki et al. (Saiki RK, et al.Proc Natl Acad Sci USA.1989, 86 (16): 6230-4), which was based on PCR- A practical improvement is made on the basis of ASO (Polymerase chain reaction&allelespecific oligonucleotide) technology. Henry Erlich, Randall Saiki and their colleagues proposed an effective improvement in the detection of HLA. This method is to label the target gene with radioactive elements during the PCR process, and then hybridize with the nylon membrane to detect the mutation of the target gene. This method can detect multiple target genes under the same conditions, thus greatly improving the detection efficiency. Subsequently, other scientists confirmed that the method can distinguish mutations of single b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/75
Inventor 程钢林炳生张帆陈华云
Owner DAAN GENE CO LTD
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