Kit for rapidly detecting nucleic acid of hepatitis C virus and detection method of kit

A hepatitis C virus and kit technology, applied in the field of biomedical detection, can solve the problems of low detection throughput, difficulty in rapid amplification, etc., and achieve the effects of high sensitivity, easy melting and denaturation, and simple operation

Active Publication Date: 2017-08-08
辽宁润基生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, some rapid quantitative PCR detection methods have appeared on the market. Among them, Roche’s Liat detection system and Cephied’s XPress are mainly realized by increasing the heating and cooling speed of the PCR instrument, that is to say, they must rely on specific fluorescence quantitative methods. Detection equipment can complete the rapid detection. Furthermore, it is difficult to achieve rapid amplification on conventional quantitative PCR instruments.
In addition, products using isothermal (or constant temperature

Method used

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  • Kit for rapidly detecting nucleic acid of hepatitis C virus and detection method of kit
  • Kit for rapidly detecting nucleic acid of hepatitis C virus and detection method of kit
  • Kit for rapidly detecting nucleic acid of hepatitis C virus and detection method of kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the preparation of kit

[0047] The kit for rapid detection of hepatitis C virus nucleic acid using one-step fluorescent quantitative RT-qPCR technology of the present invention includes nucleic acid extraction liquid, RT-qPCR reaction liquid, enzyme mixed liquid, negative control, positive control, and positive reference product.

[0048] 1. Based on the sequence information of the hepatitis C virus genome database of different genotypes, use Clustal Omega software (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to do multiple sequence comparison analysis to find different genes The conserved region of type, design the primer sequence and probe sequence that are used for the specific detection of hepatitis C virus nucleic acid, and do BLST comparison, finally artificial synthesis above-mentioned designed primer and probe, its nucleotide sequence is respectively:

[0049] Upstream primer: 5'-ACTGCTAGCCGAGTAGTGTTG-3' (SEQ ID NO.1);

[0050] Downstream primer: 5...

Embodiment 2

[0076] Embodiment 2, the use of kit

[0077] 1. Sample processing: centrifuge the blood to be tested at 1500g for 20 minutes, discard the lower blood cells, transfer the upper yellow liquid to a new storage tube, and store it at -20°C for later use.

[0078] 2. HCV RNA extraction

[0079] 1) Take 200 μl of the above samples, positive control, negative control and positive reference respectively into 1.5ml centrifuge tubes, and mark them well.

[0080] 2) Add 50 μl proteinase K and 20 μl magnetic microspheres to the centrifuge tube.

[0081] 3) Add 400 μl of RNA extraction solution I to each centrifuge tube, and incubate with shaking at room temperature for 5 minutes.

[0082] 4) Place the centrifuge tube on the magnetic stand and let it stand for 2 minutes. When the magnetic beads are completely adsorbed, carefully remove the liquid.

[0083] 5) Remove the centrifuge tube from the magnetic stand, add 600 μl RNA extraction solution II, shake and mix for 30 seconds.

[0084]...

Embodiment 3

[0096] Embodiment 3, under different temperatures, the impact of denaturant A (5%) on the amplification efficiency of hepatitis C virus nucleic acid

[0097] According to the method of using the above kit, use the hepatitis C virus nucleic acid positive sample (1000IU / ml) as a template to extract nucleic acid RNA, RT-PCR amplification, and the fluorescent quantitative PCR reaction program is only set in the third part (fast part) ) change the denaturation temperature, respectively: 95°C (swimming lane 2), 90°C (swimming lane 3), 85°C (swimming lane 4), 80°C (swimming lane 5) and 75°C (swimming lane 6). Agarose gel electrophoresis separation, staining (EB), the results are as follows figure 2 shown. It can be clearly seen from the figure that the addition of denaturing agent A (5%) can reduce the denaturation temperature, wherein, in the range of 85-95°C, the amplification can be well performed without significant difference, while at 80°C, although the denaturation temperatu...

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Abstract

The invention discloses a kit for rapidly detecting nucleic acid of a hepatitis C virus (HCV) by a one-step method. The kit comprises a nucleic acid extracting solution, an RT-qPCR reaction solution, a negative control, a positive control and a positive reference. The kit is mainly used for overcoming the disadvantage of the existing kit on the market at present that the time for nucleic acid detection is relatively long. According to the kit, the denaturation of the nucleic acid at a relatively low temperature is achieved through changing ingredients of a reaction buffer and uniquely designing and synthesizing primers and probes on the basis of the conventional RT-qPCR basic reaction principle, then, the temperature difference of denaturation and renaturation during PCR is reduced, and finally, whole reaction time is greatly shortened. The kit disclosed by the invention has relatively high detection sensitivity, specificity and repeatability and can be used for detecting common six genotypes of the hepatitis C virus; meanwhile, the kit is also applicable to the conventional quantitative PCR instrument, so that an accurate basis is provided for auxiliary diagnosis of hepatitis C virus infections and drug therapy monitoring of infected persons.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and in particular relates to a kit for rapidly detecting hepatitis C virus nucleic acid and a detection method thereof. Background technique [0002] Hepatitis C virus (Hepatitis C virus, HCV) belongs to the genus Hepatitis C virus of the family Flaviviridae. It is a spherical virus particle with a diameter of 30-60 nanometers and an envelope. . The gene coding region of HCV can be divided into two parts, the structural region and the nonstructural region, and the nonstructural region is prone to mutation. According to the heterogeneity characteristics of HCV genome, it can be divided into 6 genotypes and more than 90 different subtypes. According to the internationally accepted method, the genotype of hepatitis C virus is represented by Arabic numerals, and the gene subtype is represented by lowercase English letters. Types (such as 1a, 2b, 3a, etc.), in my country, mainly 1b and 2a genotype...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q2527/101C12Q2523/113C12Q2525/117
Inventor 王绍成康伟马南赵卓李淑君李萍苏加忱张绍峰吕志
Owner 辽宁润基生物科技有限公司
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