Method for rapidly and sensitively detecting hepatitis C virus (HCV) and genotype identification method

A hepatitis C virus, genotype technology, applied in the determination/inspection of microorganisms, microorganism-based methods, microorganisms, etc., can solve the problem of high mutation rate of RNA genome, achieve simple procedures, low demand for primers and mechanical equipment , cost-effective and easy-to-use

Inactive Publication Date: 2017-01-04
DIAGCOR LIFE SCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In addition, HCV exhibits very high heterogeneity due to the high mutation rate of its RNA genome
Traditional genotyping methods are mostly used to detect known sequences of specific genotypes, and may not be able to detect rare or emerging HCV species

Method used

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  • Method for rapidly and sensitively detecting hepatitis C virus (HCV) and genotype identification method
  • Method for rapidly and sensitively detecting hepatitis C virus (HCV) and genotype identification method
  • Method for rapidly and sensitively detecting hepatitis C virus (HCV) and genotype identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0318] Accounting expansion

[0319]This example describes an embodiment of the invention wherein the invention is used in combination with reverse transcription and PCR amplification techniques.

[0320] RNA preparation

[0321] The RNA used in the present invention can be extracted or prepared from a sample using any commercially available or appropriate method or kit.

[0322] A sample of the invention can be any type of sample, including nucleic acid from any subject or source. In one embodiment, the sample is a biological sample, such as blood, serum, plasma, saliva, sputum, semen, lymph or other bodily fluids. In another embodiment, the sample includes nucleic acid that has been roughly purified from a biological sample.

[0323] One-step RT-PCR amplification (RT-PCR)

[0324] In one embodiment, RT-PCR can be set up using the following protocol:

[0325] reaction mixture

[0326] Element Capacity (μL) HCV PCR Master Mix 13.4 IAC 0.1 ...

Embodiment 2

[0332] rapid flow hybridization

[0333] This example describes an embodiment of the invention wherein the invention is used in conjunction with flow-through hybridization techniques.

[0334] In one embodiment, the method of detecting, identifying or distinguishing HCV comprises the steps of:

[0335] a) denaturing the solution containing the target nucleic acid and contacting it with the membrane;

[0336] b) washing the membrane with a washing solution; and

[0337] c) Color developed and measured visually or spectroscopically.

[0338] In general, the detection reagents used with the present invention can be any reagent known to be useful for the detection of nucleic acids. Detection reagents include, but are not limited to, enzymes, antibodies, proteins, fluorescent chemicals, chromophores, and visual dyes. In one embodiment, the target nucleic acid is biotin-labeled during RT-PCR amplification (e.g., using biotin-labeled target-specific primers) and cross-linked by...

Embodiment 3

[0372] Validation using synthetic clones

[0373] This example illustrates one embodiment of the use of the invention to detect and discriminate nucleic acids from various HCV genotypes.

[0374] This example uses clones bearing the corresponding sequences of the 5'-UTR from various HCV genotypes to evaluate the specificity and sensitivity of the primers and probes described in this invention.

[0375] image 3 Test results of 8 synthetic clones carrying nucleic acid sequences from HCV genotypes 1, 2, 3, 4, 5 or 6 are shown. The present invention was used to amplify target nucleic acids from eight clones. The resulting amplification product is then hybridized to the probe following the procedure described above.

[0376] The test results show that the present invention can amplify and detect the target nucleic acid from all 8 clones, which is consistent with the expected result and the result of the verification control group.

[0377] Figure 4 Test results of 4 syntheti...

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Abstract

The invention provides oligonucleotides and a testing agent used for rapidly and sensitively detecting, identifying or distinguishing various genotypes and subtypes of hepatitis C viruses (HCVs). In one embodiment, the invention provides universal primers and probes corresponding to all the HCVs and the universal primers and probes can capture universal or common nucleic acids from the HCV ethnic groups. In one embodiment, the primers and the probes can capture the nucleic acids from rarely seen, unclassified or unknown HCV genotypes, subtypes or strains. In another embodiment, the invention provides probes capable of clearly detecting or identifying the HCV genotypes 1-6 or distinguishing the genotypes. In one embodiment, the invention provides primers and probes capable of clearly detecting or identifying the HCV subtypes 1a and 1b and the subtype 1a with NS3-K80 or NS3-Q80 polymorphism.

Description

technical field [0001] The present invention relates to the detection of hepatitis C virus (HCV). In one embodiment, the present invention provides methods and kits for rapid and sensitive detection and genotype identification of hepatitis C virus (HCV). Background technique [0002] Hepatitis C is a major global public health problem and one of the leading causes of chronic liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). The hepatitis C virus is usually spread through contact with the blood of an infected person. An estimated 185 million people are chronically infected with HCV, and more than 350,000 people die each year from hepatitis C-related liver disease. Hepatitis C is prevalent worldwide, with chronic HCV infection being particularly prevalent in countries including Egypt, Pakistan, and China. The virus replicates in the liver, attacks and kills liver cells, and causes live...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/707C12Q2600/106C12Q2600/156
Inventor 周国辉潘思颖李瑞萍
Owner DIAGCOR LIFE SCI LTD
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