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

A detection kit and astrovirus technology, applied in the field of rapid detection of enteric pathogens, can solve problems such as difficulty in designing viral nucleic acid molecules, and achieve the effects of improving detection sensitivity, ensuring specificity and strong specificity

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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  • Astrovirus real-time isothermal amplification detection kit, its primers and probe
  • Astrovirus real-time isothermal amplification detection kit, its primers and probe
  • Astrovirus 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 gene sequence analysis of ORF2 5′ end of astrovirus genome, astroviruses have been divided into 8 serotypes. Molecular epidemiological research data show that the most widespread astrovirus serotype worldwide is type 1. Firstly, 100 whole-genome DNA sequences of human astroviruses 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 The conserved sequence of the gene was used as a candidate region for primer and probe design, and the primer and probe design was carried out in combination with the software Oligo 7.56 (Molecular Biology Insights, Inc. USA). The design idea is to comprehensively consider the specificity and generality of the detection of astroviruses (degenerate bases are ...

Embodiment 2

[0042] Example 2: Establishment and optimization of astrovirus 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 performed using in vitro transcribed RNA from a part of the genome fragment of astrovirus 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 / 1.6mM,...

Embodiment 3

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

[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 genomic RNA fragment transcribed in vitro by Astr...

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PUM

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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 an Astrovirus 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 an Astrovirus ORF2 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 stellate real-time NASBA isothermal amplification rapid detection kit; it also relates to a pair of primers and a molecular beacon probe specific to the astrovirus genome used in the kit. Background technique [0002] Astrovirus (Astrovirus, AstV) was first discovered in 1975 by Appleton and Higgins when they used electron microscopy to detect stool samples from children with acute gastroenteritis. Metamorphosis was named Astrovirus by Madeley and Cosgrove. Astrovirus is widely distributed and is one of the important pathogens that cause acute diarrhea in infants, the elderly, and immunocompromised or deficient persons. Astroviruses can cause sporadic and iatrogenic infections, as well as outbreaks, and some studies have fo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 莫秋华谭华李卫岗杨泽刘志明林继灿杜田
Owner 珠海国际旅行卫生保健中心
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