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A highly specific probe for real-time detection of nasba products

A probe and specific technology, applied in the field of high-specific probes for real-time detection of NASBA products, can solve the problems of difficult diagnosis, non-specific reaction, high concentration, etc., and achieve the effect of shortening the detection time, strong specificity and high sensitivity

Active Publication Date: 2019-12-10
CAPITALBIO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used method for on-line detection of NASBA amplification products is the molecular beacon method. However, since the NASBA amplification reaction is combined by three enzymes, and the concentration of the three enzymes is relatively high, they are highly aggressive to the nucleic acid in the system. The extension of the molecular beacon in the system makes the primers prone to non-specific reactions, resulting in the generation of false positive signals, resulting in misjudgment of results, and bringing certain difficulties to subsequent diagnosis.

Method used

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  • A highly specific probe for real-time detection of nasba products
  • A highly specific probe for real-time detection of nasba products
  • A highly specific probe for real-time detection of nasba products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Embodiment 1, the adjacent probe that is used for the detection of influenza virus NASBA amplification product

[0029] The present invention designs four sets of primers and adjacent probes for the detection of influenza virus NASBA amplification products, the primers used for influenza A virus gene detection are InfA-TY-PrimerF, InfA-TY-PrimerR, and the corresponding adjacent probes are InfA -TY-ProbeF, InfA-TY-ProbeR, the primers used for the detection of influenza A virus H1 subtype gene are InfA-H1-PrimerF, InfA-H1-PrimerR, and the corresponding adjacent probes are InfA-H1-ProbeF, InfA- H1-ProbeR, the primers used for the detection of influenza A virus H3 subtype genes are InfA-H3-PrimerF, InfA-H3-PrimerR, and the corresponding adjacent probes are InfA-H3-ProbeF, InfA-H3-ProbeR, which are used for B The primers for influenza virus gene detection are InfB-TY-PrimerF, InfB-TY-PrimerR, and the corresponding adjacent probes are InfB-TY-ProbeF, InfB-TY-ProbeR. The NASBA...

Embodiment 2

[0035] Embodiment 2, adjacent probe is used for NASBA product detection

[0036] 1. Preparation of RNA template

[0037] The plasmids used to prepare RNA templates were provided by Boao Bio Group Co., Ltd., including recombinant plasmid pUC-InfA-TY containing influenza A virus gene, recombinant plasmid pUC-InfA-H1 containing influenza A virus H1 subtype HA gene, containing The recombinant plasmid pUC-InfA-H3 containing the HA gene of influenza A virus H3 subtype, and the recombinant plasmid pUC-InfB-TY containing the gene of influenza B virus. The RNA template preparation process is as follows.

[0038] 1. Enzyme digestion: first digest each recombinant plasmid to be tested with EcoRI endonuclease at 37°C for 2 hours.

[0039] 2. Transcription: Prepare a reaction with a total volume of 50 μL by 5 μL 5×Transcription Optimized Buffer (Promega), 2U T7 RNA polymerase, 10 mM DTT (Promega), 10 U recombinant RNase inhibitor (Promega), 2 mM rNTP, and 5 μL of digested product system...

Embodiment 3

[0060] Embodiment 3, adjacent probe characteristic analysis

[0061] 1. Concentration of adjacent probes

[0062] 1. Preparation of constant temperature amplification system with different adjacent probe concentrations

[0063] Isothermal amplification buffer B1: 200mM Tris-HCL (pH 8.0), 0.5μM InfA-TY-PrimerF, 0.5μM InfA-TY-PrimerR, 0.01μM InfA-TY-ProbeF, 0.01μM InfA-TY-ProbeR, 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2 , 450mM KCl, 15% by volume DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.

[0064] Isothermal amplification buffer B2: 200mM Tris-HCL (pH 8.0), 0.5μM InfA-TY-PrimerF, 0.5μM InfA-TY-PrimerR, 0.1μM InfA-TY-ProbeF, 0.1μM InfA-TY-ProbeR, 50mM DTT, 10mM dNTPs, 10mM rNTPs, 80mM MgCl 2 , 450mM KCl, 15% by volume DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.

[0065] Isothermal amplification buffer B3: 200mM Tris-HCL (pH 8.0), 0.5μM InfA-TY-PrimerF, 0.5μM InfA-TY-PrimerR, 1μM InfA-TY-ProbeF, 1μM InfA-TY-ProbeR, 50mM DTT, 10mM dNTP , 10mM rNTP, 80m...

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Abstract

The invention belongs to the technical field of nucleic acid amplification, and relates to a highly specific probe for real-time detection of NASBA (Nucleic Acid Sequence Based Amplification) products and a method for detecting the NASBA products. The highly specific probe comprises two adjacent nucleotide sequences, bases from the first position to the fourth position at the 3' end of each of the two adjacent nucleotide sequences are ribonucleotide bases, and the remaining bases are all deoxyribonucleotide bases. The highly specific probe is applied to detection of the NASBA products, has high sensitivity and specificity, and reduces detection time, thereby enabling the NASBA detection technology to be better applied to detection of nucleic acid of pathogenic microorganisms.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification and relates to a high-specificity probe for real-time detection of NASBA products. Background technique [0002] In recent years, with the rapid development of molecular biology technology, a large number of nucleic acid detection-based diagnostic methods have been established and widely used in molecular diagnostic reagents. Constant temperature amplification technology is a new in vitro nucleic acid amplification technology developed after PCR technology. At present, the constant temperature amplification techniques mainly include: rolling circle nucleic acid amplification, loop-mediated isothermal amplification, strand displacement amplification, nucleic acid sequence-dependent amplification, and helicase amplification. Isothermal amplification has the advantages of rapidity, high efficiency, and good specificity, and does not require special equipment. [0003] Nucleic Aci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6865C12N15/11
CPCC12Q1/6865C12Q2525/121C12Q2563/107
Inventor 张岩盖伟邢婉丽单万水刘厚明宋翠丹程京
Owner CAPITALBIO CORP
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