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A method of fluorescent detection of isothermal loop-mediated amplification (LAMP) of a target nucleic acid, oligonucleotides and kits thereof

A technology of oligonucleotide and target nucleic acid sequence, applied in the direction of microbial determination/inspection, biochemical equipment and methods, recombinant DNA technology, etc. Sexual nucleotide sequence and other issues

Inactive Publication Date: 2018-08-21
DIASORIN ITALIA SPA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this strategy has the disadvantage of being dependent on the specific nucleotide sequence of the target nucleic acid and is limited by the presence and / or position of guanine nucleotides within this sequence.

Method used

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  • A method of fluorescent detection of isothermal loop-mediated amplification (LAMP) of a target nucleic acid, oligonucleotides and kits thereof
  • A method of fluorescent detection of isothermal loop-mediated amplification (LAMP) of a target nucleic acid, oligonucleotides and kits thereof
  • A method of fluorescent detection of isothermal loop-mediated amplification (LAMP) of a target nucleic acid, oligonucleotides and kits thereof

Examples

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Effect test

Embodiment 1

[0051] Example 1 - Comparison of the LAMP method of the present invention with the LAMP assay of Chou et al.

[0052] Sample Preparation

[0053] To compare the LAMP method of the present invention with the LAMP assay described by Chou et al., appropriate target nucleic acid sequences were prepared. Briefly, a 350-bp DNA fragment derived from the white spot syndrome virus (WSSV) genome (nt226681-227934, GenBank AF332093.1) was cloned into the pMA-T vector by using a combination of Sfi I / Sfi I restriction sites (GENEART) to provide a positive control. 2×10 was prepared for the recombinant WSSV plasmid 6 copies / μL to 2×10 1 10-fold dilution series of copies / μL.

[0054] Two different plasmid dilution series were prepared and used as Tris-HCl 10 mM, pH 8.5 alone, or this buffer containing human genomic DNA (20 ng / μL) in addition. In this study, analyzed plasmid dilutions were denatured at 100°C for 10 min. Immediately after denaturation, plasmid samples were placed on i...

Embodiment 2

[0069] Example 2 - Comparison of the LAMP method of the present invention with an intercalating dye based LAMP assay.

[0070] Sample Preparation

[0071] In order to compare the performance of the methods of the invention with LAMP assays involving the use of intercalating dyes, appropriate target nucleic acid sequences were prepared. Briefly, a positive control was provided by cloning a 350-bp DNA fragment derived from the MYH11 gene (GenBank D10667.1) into the pMA-T vector (GENEART) using the Sfi I / Sfi I restriction site combination.

[0072] The recombinant MYH11 plasmid was serially diluted 10-fold in buffer Tris-HCl 10 mM, pH 8.5, from approximately 2 × 10 6 copies / μL to 2×10 1 copies / μL.

[0073] In this study, assayed plasmid dilutions were denatured at 100°C for 10 min. Immediately after denaturation, plasmid samples were placed on ice for 10 min.

[0074] LAMP reaction

[0075] The LAMP oligonucleotide primers and probes used in the comparative analysis a...

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Abstract

The invention concerns a method for detecting isothermal loop-mediated (LAMP) amplification of a target nucleic acid sequence which is based on the fluorescence resonance energy transfer (FRET) mechanism. The invention also concerns a set of oligonucleotides and a kit adapted for carrying out the LAMP-FRET method of the invention.

Description

technical field [0001] The present invention relates to methods for detecting nucleic acid amplification by loop-mediated isothermal amplification (LAMP), and a set of oligonucleotides and kits for performing the methods of the invention. Background technique [0002] Loop-mediated isothermal amplification (LAMP) is a recently developed method of nucleic acid amplification via an automated cyclic strand displacement reaction, usually performed at a constant temperature of 60°C to 65°C (Notomi T. et al 2000. Nucleic acids Res. Vol. 28(12)-e63). This technique employs a DNA polymerase with strand-displacing activity and a set of four oligonucleotides, called inner and outer primers, specifically designed to recognize six different recognition sites on a target nucleic acid. The two outer primers only play a role in strand displacement during the acyclic step, while the inner primer includes both sense and antisense sequences and helps to form a typical LAMP amplification prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6853C12N15/11
CPCC12Q1/6853C12Q1/6844C12Q2531/119C12Q2565/101C12Q2525/161C12Q2525/301C12Q2527/101C12Q2549/119
Inventor 久利亚·米奴茨里卡尔多·麦斯特里尼
Owner DIASORIN ITALIA SPA