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Group A rotavirus real-time isothermal amplification detection kit, primers and probe thereof

A group A rotavirus, detection kit technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc. High efficiency and specificity

Active Publication Date: 2013-03-13
SHENZHEN TOTAL TEST TECH
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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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  • Group A rotavirus real-time isothermal amplification detection kit, primers and probe thereof
  • Group A rotavirus real-time isothermal amplification detection kit, primers and probe thereof
  • Group A rotavirus real-time isothermal amplification detection kit, primers and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0032] Rotaviruses are divided into different groups according to different serological responses, and each group can be divided into different serotypes. So far, seven rotavirus groups (groups A to G) have been reported. Group A rotavirus is the main pathogen of severe diarrhea in infants and young children. The rotavirus group antigen is determined by the VP6 gene, while the VP7 and VP4 genes determine the G and P serotypes, respectively. Therefore, selecting the highly conserved nucleic acid sequence of the VP6 gene as the target sequence for real-time NASBA primer and probe design is representative for the detection of group A rotaviruses. Firstly, 100 DNA sequences of group A rotavirus VP6 genes were downloaded from the gene bank of NCBI in the United States, and then the homologous comparison analysis was performed on the downloaded sequences using the molecular biology software DNAMAN 6.0 (Lynnon Corpo...

Embodiment 2

[0042] Example 2: Establishment and optimization of a real-time NASBA detection system for group A rotavirus

[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 the in vitro transcribed RNA of group A rotavirus VP6 gene 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, 0...

Embodiment 3

[0051] Example 3: Composition and detection method of real-time NASBA kit for detecting group A rotavirus

[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 (sorbitol);

[0055] (3) Positive control: VP6 gene RNA fragment transcribed in vitro b...

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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 group A rotavirus real-time isothermal amplification detection kit, a pair of primers and a molecular beacon probe. 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 group A rotavirus VP6 gene. 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. Specifically, it relates to a real-time NASBA isothermal amplification rapid detection kit for group A rotavirus; it also relates to a pair of primers and a molecular beacon probe specific for group A rotavirus VP6 gene used in the kit. Background technique [0002] Group A rotavirus (Rotavirus, RV) is the most important pathogen causing severe diarrhea in infants and young children worldwide, accounting for more than 50% of the etiology of pediatric intestinal infections. Its genome consists of 11 discontinuous double-stranded RNA gene segments, Among them, the antigenic difference of the structural protein encoded by the VP6 gene is the basis for grouping rotaviruses. Group A rotavirus is also one of the main causes of infant mortality in developing c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 莫秋华伍碧梅赵俊华杨泽谭华林继灿杜田
Owner SHENZHEN TOTAL TEST TECH
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