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Norovirus real-time isothermal amplification detection kit, its primers and probe

A detection kit and virus detection technology, applied in biochemical equipment and methods, microorganisms, and methods based on microorganisms, etc., can solve problems such as the difficulty in designing viral nucleic acid molecules, and achieve improved detection sensitivity, guaranteed specificity, and amplification efficiency high effect

Inactive Publication Date: 2013-03-13
珠海国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Loop-mediated Isothermal Amplification of DNA (LAMP) technology is widely used, and there are related patent applications. However, LAMP technology requires the use of 4 primers to identify 6 different regions of the target DNA. Although Improved specificity, but very difficult to design for highly variable viral nucleic acid molecules

Method used

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  • Norovirus real-time isothermal amplification detection kit, its primers and probe
  • Norovirus real-time isothermal amplification detection kit, its primers and probe
  • Norovirus real-time isothermal amplification detection kit, its primers and probe

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Design of real-time NASBA primers and probes

[0032] According to the differences in the RdRp and capsid protein nucleotide sequences of the NVs genome, NVs can be divided into five genetic groups (Genogroup), among which Genogroup I (Genogroup I, GGI) and Genogroup II (Genogroup II, GGII) and Temporary The designated genetic group IV (Genogroup IV, GGIV) infects humans. Genetic groups GI and GII can be divided into several genotypes. Epidemiological data show that GGII / 4 NV is the most common genotype in the world, and the NV infected by patients in my country is also mainly genotype GGII / 4. Firstly, 100 complete genome DNA sequences of GII Norovirus were downloaded from the NCBI gene bank in the United States, and then the homology comparison analysis was performed on the downloaded sequences using the molecular biology software DNAMAN 6.0 (Lynnon Corporation, Quebec, Canada) to find highly homologous sequences. The conserved sequence of the source was...

Embodiment 2

[0042] Example 2: Establishment and optimization of norovirus real-time NASBA detection system

[0043] The conditions were optimized for some important influencing factors of the real-time NASBA detection system.

[0044] 1. Method

[0045] (1)K + Concentration: prepare 2× real-time NASBA reaction solution according to the reaction system in Table 1, fix other parameters, and adjust K respectively + The final concentration was 40mM, 60mM, 80mM, 100mM, 120mM, 240mM, and real-time NASBA isothermal amplification was carried out using the in vitro transcribed RNA of the partial gene fragment of Norovirus ORFs1 as a template. After the experiment, compare different K + Effect of concentration on amplification efficiency and fluorescence curve.

[0046] (2) Concentration combination of dNTP / NTP: Prepare 2× real-time NASBA reaction solution according to the reaction system in Table 1, fix other parameters, and adjust the final concentration of dNTP / NTP to 0.1mM / 1.6mM, 0.2mM / 1....

Embodiment 3

[0051] Embodiment 3: Composition and detection method of real-time NASBA kit for detecting Norovirus

[0052] 1. Composition of the kit (stored at -20°C)

[0053] (1) 2× real-time NASBA reaction solution: its components are: 120mM Tris-HCl (pH8.0), 240mM KCl, 20mM MgCl2, 24mM DTT, 4mM spermidine, 0.4mM dNTP, 3.2mM NTP, 0.3mM trehalose, 0.4mM Betaine, 30% DMSO, 0.8μM Forward Primer F1, 0.8μM Reverse Primer R1, 0.4μM Molecular Beacon Probe P1; wherein the forward primer F1 is the nucleus shown in SEQ ID NO:1 Nucleotide sequence, the reverse primer R1 is the nucleotide sequence shown in SEQ ID NO: 2, and the molecular beacon probe P1 is the nucleotide sequence shown in SEQ ID NO: 3;

[0054] (2) 5×enzyme mixture: its components are: 1.5 U / μL AMV reverse transcriptase, 6 U / μL T7 RNA transcriptase, 0.0625 U / μL RNAase H enzyme, 5 U / μL Ribonuclease Inhibitor, 20 mg / mL BSA, 8 mM sorbitol;

[0055] (3) Positive control: a partial gene fragment of ORFs1 transcribed in vitro by nor...

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Abstract

The invention relates to an enteropathogen rapid detection technology based on real-time nucleic acid sequence-based amplification (NASBA). Specifically, the invention provides a Norovirus real-time isothermal amplification detection kit, and a pair of primers and a molecular beacon probe thereof. The kit includes: a 2*real-time NASBA reaction solution containing the primers and the probe, a 5*enzyme mixed solution and a positive control template, a negative control and a blank control. The sequences of the primers and the probe are the sequences numbered as SEQIDNO:1-3, and the primers and the probe can specifically amplify and detect a Norovirus ORFs1 gene fragment. The kit provided in the invention has the characteristics of fastness, high efficiency, sensitivity and specificity, and real-time detection analysis, etc., and can be used in the fields of conventional detection and disease control and prevention in clinical practice and ports.

Description

technical field [0001] The invention relates to a rapid detection technology of intestinal pathogens based on real-time nucleic acid sequence-based amplification (Nucleic acid sequence-based amplification, NASBA) technology. It specifically relates to a norovirus real-time NASBA isothermal amplification rapid detection kit; it also relates to a primer pair and a molecular beacon probe specific for norovirus genome used in the kit. Background technique [0002] Norovirus (NV), also known as Norwalk-like virus, represented by Norwalk virus, is a widely distributed virus that can cause acute gastroenteritis in adults and children. The most important viral pathogen causing diarrhea other than rotavirus is closely related to the outbreak of acute gastroenteritis caused by food and water pollution. The norovirus genome is a single-stranded positive-sense RNA with a full length of 7642 nt, including three open reading frames (open reading frames, ORFs) ORFs1, ORFs2 and ORFs3. ORF...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 莫秋华冯子力李卫岗杨泽林继灿刘志明杜坚
Owner 珠海国际旅行卫生保健中心
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