Oligonucleotides chip composition for analyzing HCV gene type and its detecting method

The technology of oligonucleotide probe and composition is applied in the field of oligonucleotide chip composition for analyzing hepatitis C virus genotype and its detection field, which can solve the problems of time-consuming and labor-intensive processing and analysis, etc.

Inactive Publication Date: 2004-10-06
株式会社生物核心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to this method, precise results can be obtained with several probes, but immobilizing many probes on the NC membrane has a certain limit
In addition, this method takes a lot of time and labor to process and analyze various samples because only one genotype of a person can be analyzed on this NC membrane

Method used

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  • Oligonucleotides chip composition for analyzing HCV gene type and its detecting method
  • Oligonucleotides chip composition for analyzing HCV gene type and its detecting method
  • Oligonucleotides chip composition for analyzing HCV gene type and its detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Artificial synthesis and base sequence of HCV primers

[0021] HCV PCR primers as shown in Table 1 were used for RT-PCR by analyzing the sites that co-react with the type 6 5'UTR. Antisense primers for secondary asymmetric PCR were classified into two categories according to the method of identification. In the first category, primers are attached to biotin to identify chromogenic reactions by hybridization. In the second class, anthocyanins were used to identify fluorescent reactions through hybridization reactions. An additional 25 bp base (SP6) was artificially synthesized at the 5' end of the secondary PCR antisense primer, and a base complementary to this site linked to anthocyanin was synthesized. The primers were synthesized by MWG-biotech company (Germany) according to the inventor's reservation, and the primers were synthesized by the method for synthesizing oligonucleotides written in Section 10.42 of the third edition of "Molecular Cloning" (Sa...

Embodiment 2

[0023] Embodiment 2: the preparation of HCV RNA, reverse transcription-primary PCR and secondary PCR reaction

[0024] 1) Mix 5 ul of HCV RNA extraction buffer (DEPC-DW 860 ul in 1 ml, Taq 10×buffer 100 ul, 1M DTT 20 ul, 10% NP40 20 ul) and 10 ul of plasma or serum.

[0025] 2) Heat the test tube containing the mixed serum in the PCR equipment at 92°C for 2 minutes, then quickly tap the test tube on ice

[0026] 3) Centrifuge the test tube for 5-10 seconds

[0027] 4) Then carry out reverse transcription and primary PCR reaction in GeneAmp PCR system 9600 thermal cycler (Perkin ElmerCetus, U.S.A) as shown in Table 2

[0028] 5) Carry out secondary asymmetric PCR reaction with 2ul primary PCR product as shown in Table 3

[0029] 6) Mix 1 ul of gel loading buffer (0.25% bromophenol blue, 0.25% xylidine FF, 15% non-polysucrose 400) into 5 ul of the secondary asymmetric PCR product, and add 1 μg / ml of bromine After electrophoresis on 2% agarose gel with ethidium (EtBr), a ban...

Embodiment 3

[0032] Embodiment 3: be used for the synthesis and base sequence of the probe of preparation HCV oligonucleotide chip

[0033] All probes have an amino linkage added to the 5' end for covalent attachment to acetaldehyde glass. 10-20 oligo(dT) are attached to the probe to facilitate the hybridization reaction. Then, the base sequence shown in Table 5 was ligated to amino-linked-oligo(dT) 10-20 . Short representation, with "amino linkage - oligo(dT) 10-20 -The primers of the "probe base sequence" arrangement order are synthesized in MWG-biotech company (Germany) according to the inventor's reservation. The base sequences of the identified HCV genotypes are analyzed as shown in Table 4 and belong to 6 types. HCV genes from 53 species were identified.

[0034] HCV type

HCV category

1a

HCV-1(M62321), HCV-H(M67463), HC-J1(D10749), GM1(M61728),

GM2(M61719), H90(M62382), US9(L38353), H77(AF009606), H99, PT-1

1b

HCV...

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Abstract

The invention discloses a method for analyzing HCV genotype by extracting RNA from plasma and serum, carrying out RT-PCR of hepatitis C virus (HCV) and reacting it on an oligonucleotide chip. The present invention also provides a simple and accurate method for assaying 4 human HCV genotypes on one slide.

Description

technical field [0001] The invention relates to an oligonucleotide chip composition and a manufacturing method thereof, in particular to an oligonucleotide chip composition for analyzing the genotype of hepatitis C virus (HCV) and a detection method thereof. technical background [0002] In general, hepatitis C virus (hereinafter referred to as 'HCV'), a type of hepatitis virus, is a major factor causing serious diseases such as hepatitis including acute hepatitis and chronic hepatitis, which may develop into cirrhosis and hepatoma . HCV is transmitted by blood transfusion and body fluids (Choo et al., Science 244, 359-362, 1989). It is estimated that there are about 400 million people infected with HCV in the world: 0.2-2% people in developed countries such as Europe, North America and Japan; 2-5% in South America and Asia; more than 5% in Africa; 1.6% in South Korea (Park et al., J. Viral Hepat. 2, 195-202, 1995). HCV is a very dangerous virus for human health, and unli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/68C12Q1/70
CPCC12Q1/707
Inventor 朴荣石朴宰赞金银夏
Owner 株式会社生物核心
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