Kit stored at 2 to 8 DEG C and used for rapidly detecting hepatis c virus nucleic acid

A hepatitis C virus and kit technology, which is applied in the determination/inspection of microorganisms, microorganism-based methods, microorganisms, etc., can solve the problems of high requirements for reagent transportation conditions, poor thermal stability of reverse transcriptase, and prolonged detection time. Achieve the effect of improving convenience, wide linear range and reducing time

Inactive Publication Date: 2018-12-11
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the poor thermal stability of reverse transcriptase, the detection reagents are usually stored at -20°C in a solid state, and the reagents must be thawed before use, which is inconvenient to operate and prolongs the detection time. The detection performance is affected to a certain extent; and the frozen storage reagents have high requirements for transportation conditions, which further increases the detection cost
Currently on the market, only Roche’s hepatitis C virus nucleic acid detection reagent is stored in a liquid state at 2-8°C. Although the operation is more convenient, it takes 4.5 hours to complete nucleic acid extraction and nucleic acid amplification detection, which is time-consuming.
So far, there is no HCV nucleic acid detection reagent that can be stored at 2-8°C and has rapid detection capabilities.

Method used

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  • Kit stored at 2 to 8 DEG C and used for rapidly detecting hepatis c virus nucleic acid
  • Kit stored at 2 to 8 DEG C and used for rapidly detecting hepatis c virus nucleic acid
  • Kit stored at 2 to 8 DEG C and used for rapidly detecting hepatis c virus nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 The composition of kit of the present invention

[0040]1. 20ulqRT-PCR reaction solution: 5%DMSO, 300mM betaine, 0.05%Tween20, 5% glycerol, 50mM tris(hydroxymethyl)methylglycine (pH8.3), 100mM potassium acetate, 0.6uM primer 1, 0.6 uM Primer 2, 0.2uM Primer 3, 0.2uM Primer 4, 0.3uM Probe 5, 0.1uM Probe 6, 10U rTth DNA Polymerase, 0.5U UNG Enzyme, 0.2mMdATP, 0.2mMdGTP, 0.2mMdCTP, 0.4mMdUTP, 0.1 % sodium azide; 4 specific primers and 2 specific probes are:

[0041] Primer 1 (SEQ ID No.1): 5'-GTCTAGCCATGGCGTTAGTA-3';

[0042] Primer 2 (SEQ ID No.2): 5'-AAGCACCCTATCAGGCAGTA-3';

[0043] Primer 3 (SEQ ID No.3): 5'-GGCTGCTCGCGGATACCC-3';

[0044] Primer 4 (SEQ ID No.4): 5'-CTGAAGAACTTGCGTTCTCG-3';

[0045] Probe 1 (SEQ ID No.5): FAM-CGGAACCGGTGAGTACACCGGAAT-BHQ1;

[0046] Probe 2 (SEQ ID No. 6): ROX-ACCTCGGGTTTCCGTCTTGCTCGT-BHQ2.

[0047] 2. 10ulqRT-PCR starter: 1.5mM Mn(OAc) 2 and 0.1% sodium azide composition.

[0048] 3. RNA internal control: RNA pseud...

Embodiment 2

[0054] Example 2 Extraction of Hepatitis C Virus Nucleic Acid

[0055] In this example, when extracting and purifying the hepatitis C virus nucleic acid in the sample, clinical serum or plasma samples and the nucleic acid extraction and purification reagent (magnetic bead method) of Zhengzhou Antu Bioengineering Co., Ltd. were used, and the specific operation method was carried out according to the instructions.

Embodiment 3

[0056] Embodiment 3 The minimum detection limit of the kit of the present invention

[0057] Dilute the WHO quantitative standard (WHO International Standard 5th WHO International StandardforHCV NAT NIBSC code: 14 / 150) with negative plasma to 15, 10, 7.5, 5, 2.5IU / ml, using nucleic acid from Zhengzhou Antu Bioengineering Co., Ltd. The extraction and purification reagent (magnetic bead method) was used to extract and purify hepatitis C virus nucleic acid, and this kit was used for detection. Each concentration sample was repeated 60 times, and the detection rate of each concentration sample was calculated to determine the minimum detection limit ( The lowest concentration with a detection rate ≥ 95% was defined as the lowest detection limit), and the results are shown in Table 1. It can be seen from the results that the minimum detection limit of this kit is 10IU / ml.

[0058] Table 1

[0059]

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Abstract

The invention discloses a kit stored at 2 to 8 DEG C and used for rapidly detecting hepatis c virus nucleic acid. The kit comprises a qRT-PCR reaction solution, a qRT-PCR initiator, an RNA internal control, a strongly positive quality control product, a weakly positive quality control product, a negative quality control product and a quantitative reference product; and the qRT-PCR reaction solution comprises a qRT-PCR reinforcing agent, a reaction buffer solution, two specific primers for detecting the hepatis c virus nucleic acid, one specific probe for detecting the hepatis c virus nucleic acid, two specific primers for detecting the RNA internal control, one specific probe for detecting the RNA internal control, a bi-directional DNA polymerase, UNG enzyme, dATP, dGTP, dCTP, dUTP and 0.1percent sodium azide. The kit is high in sensitivity (the minimum detection limit of the HCV virus is 10IU/ml), good in specificity, high in precision, accurate in qualification, and wide in linear range, simple in detection method, short in detection time, high in reference value for clinically and rapidly detecting the hepatis c virus nucleic acid, and suitable for popularization and application.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to a kit for rapidly detecting hepatitis C virus nucleic acid stored at 2-8°C. Background technique [0002] Hepatitis C virus (Hepatitis C Virus, HCV) belongs to the Flaviviridae family and is a single-stranded positive-sense RNA virus. Currently, it can be divided into 6 genotypes and multiple subtypes. The hepatitis C viral hepatitis caused by HCV infection is global Popularity. Chronic viral hepatitis C can lead to chronic inflammation, necrosis and fibrosis of the liver, and some patients may develop liver cirrhosis or even hepatocellular carcinoma, which is extremely harmful to the health and life of patients and has become a serious social and public health problem. The nucleic acid detection of hepatitis virus provides an effective method for the clinical diagnosis and treatment of hepatitis C. Currently, nucleic acid detection methods for HCV are mainly based o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/707C12Q2531/113C12Q2527/156C12Q2545/101C12Q2545/113
Inventor 王义聪李振红杜美鲁清月付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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