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Hepatitis c virus genotype detection kit

A technology of hepatitis C virus and detection kit, which is applied in the direction of microorganism-based methods, detection/testing of microorganisms, microorganisms, etc., and can solve the problems of cumbersome operation, low detection rate of specimens, incomplete coverage, etc.

Active Publication Date: 2014-04-16
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many deficiencies in this type of kit: 1) The detection sensitivity is low, about 10000IU / ml; the detection range is narrow, generally between 1.00E+04IU / ml~1.00E+07IU / ml. Samples with greater than 5.00E+07IU / ml) and low values ​​(less than 1.00E+04IU / ml) cannot be detected; 2) For HCV genotype coverage is not complete, only subtypes under one of the genotypes can be detected; 3) Phenol - The chloroform method is the most classic RNA extraction method, but it is cumbersome to operate, requires high equipment and personnel operations, the detection rate of samples with low viral load is low, and the reagents used have certain toxicity; the column extraction method does not require high-speed centrifugation, but Frequent replacement of centrifuge tubes is required, which takes a long time and has poor specificity; 4) PCR inhibitors in samples cannot be effectively removed (such as blood lipids, strong hemolysis, etc.); Widely carried out

Method used

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  • Hepatitis c virus genotype detection kit
  • Hepatitis c virus genotype detection kit
  • Hepatitis c virus genotype detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1: provide a kind of HCV genotype detection kit

[0091] A HCV genotype detection kit, which at least consists of the following independent components:

[0092] HCV1 PCR reaction solution: 10 μl of 5×PCR reaction buffer (included with Tth polymerase), 0.2 mmol / L deoxyribonucleoside triphosphate, 0.2 μmol / L~0.4 μmol / L for target polynucleotide amplification The upstream and downstream primers HCV1-F and HCV1-R, the probe HCV1-P for target polynucleotide detection at 0.2 μmol / L to 0.4 μmol / L, and the upstream and downstream primers for target polynucleotide amplification And the probes used for target polynucleotide detection, the base pair sequences are respectively:

[0093] Upstream primer HCV1-F: 5'-AGGAAGACTTCCGAGCGGTC-3';

[0094] Downstream primer HCV1-R: 5'-TGCCATAGAGGGGCCAAGG-3';

[0095] Probe HCV1-P: 5'FAM-TACCCGGGCTGCGCCCAGG-BHQ13';

[0096]HCV2 type PCR reaction solution: 10 μl of 5×PCR reaction buffer (included with Tth polymerase), 0.2 mmol / L...

Embodiment 2

[0116] Embodiment 2: provide a kind of HCV genotype detection kit

[0117] A kind of HCV genotype detection kit, it also contains following several independently existing components except containing each component existing independently in embodiment 1:

[0118] HCV typing enzyme mixture: Tth enzyme 5U / μl~15U / μl, 1U / μl~10U / μl H-Taq DNA polymerase;

[0119] HCV typing positive control: calibrate the pseudovirus of known concentration, the concentration is 1.00~5.00E+05IU / ml.

[0120] HCV typing negative control: sterile saline.

Embodiment 3

[0121] Embodiment 3: provide a kind of HCV genotype detection kit

[0122] A HCV genotype detection kit, which at least consists of the following more than a dozen independently existing components:

[0123] RNA extraction solution I: 0.2% to 1.0% (mass / volume) of sodium lauryl sulfate, triton

[0124] 1.0%~4.0% (volume / volume), guanidine isothiocyanate 0.2mol / L~1.0mol / L, 100~400μg / ml magnetic beads;

[0125] RNA extraction solution II: 4-hydroxyethylpiperazineethanesulfonic acid 100-300mmol / L, pH6.5±0.2, sodium chloride 100-300mmol / L;

[0126] RNA extraction solution III: Triton 0.1%~1.0% (volume / volume), sodium chloride 100~300mmol / L;

[0127] RNA extraction solution IV: mineral oil;

[0128] RNA eluent: Tris-HCl0.8~1.2mol / L, EDTA0.1~1.0mol / L

[0129] HCV1 PCR reaction solution: 10 μl of 5×PCR reaction buffer (included with Tth polymerase), 0.2 mmol / L deoxyribonucleoside triphosphate, 0.2 μmol / L~0.4 μmol / L for target polynucleotide amplification The upstream and downstr...

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Abstract

The invention relates to a hepatitis c virus (HCV) genotype detection kit. The kit utilizes a paramagnetic particle method to extract sample nucleic acid, and by adopting a real-time fluorescence quantitative PCR (polymerase chain reaction) technology, taking a highly conserved area of an HCV genome as an amplification target, and designing a specificity primer and a TaqMan probe, typing detection of the HCV gene can be rapidly and accurately carried out on a real-time fluorescence PCR instrument through PCR amplification.

Description

technical field [0001] The invention belongs to the technical field of hepatitis C virus genotype detection and relates to a hepatitis C virus genotype detection kit. Background technique [0002] The magnetic bead method is a nucleic acid extraction method that has developed rapidly and is widely used in recent years. Its advantages over traditional methods can be summarized as follows: large variability in extraction volume, strong specificity of adsorbed nucleic acids, high purity, and realizable Automate operations. [0003] Real-time fluorescent PCR technology (FQ-PCR) integrates PCR, molecular hybridization and photochemistry, so that the whole process of PCR amplification and product analysis is carried out under single-tube closed conditions. Compared with other detection technologies, real-time fluorescent PCR technology has the following advantages: (1) Compared with immunological detection, it has higher sensitivity. Theoretically, only one gene copy can be detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/70C12Q2561/113C12Q2561/101C12Q2531/113
Inventor 戴立忠黄河刘佳
Owner SANSURE BIOTECH INC
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