Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

a nucleic acid and fluorophore technology, applied in the field of nucleic acid probes, can solve the problems of difficult to distinguish between specific and unspecific dna products generated under isothermal conditions, and the commonly used probes such as taqman® probes are not compatible with lamp technology, so as to achieve specificity of dna products amplified in isothermal reaction

Inactive Publication Date: 2016-09-22
MAST GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009]The cytosine base is preferably substantially centrally disposed along the oligonucleotide's length. There are particular benefits associated with labeling the probe internally at a cytosine base. The specificity of the DNA product amplified in an isothermal reaction may be confirmed using a melt curve analysis. However due to a large number of product variants generated in this reaction and a low resolution of melt curve analysis, using intercalating dyes like V13, it is very difficult to distinguish between specific and unspecific DNA products generated under isothermal conditions. Commonly used probes such as TaqMan® probe are not compatible with LAMP technology due to the strand displacement activity of BST polymerase. The probe of the invention is elongated and becomes incorporated into a DNA product during isothermal amplification, which allows for performing a melt curve analysis on the generated product. In the probe of the invention, the fluorophore is conjugated to an internal cytosine complementary to guanine in the antisense strand. Guanine affects the excitation state of many fluorophores resulting in a formation of unique melt curve signatures and allows distinguishing between specific and unspecific products generated under isothermal conditions.

Problems solved by technology

However due to a large number of product variants generated in this reaction and a low resolution of melt curve analysis, using intercalating dyes like V13, it is very difficult to distinguish between specific and unspecific DNA products generated under isothermal conditions.
Commonly used probes such as TaqMan® probe are not compatible with LAMP technology due to the strand displacement activity of BST polymerase.

Method used

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  • Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification
  • Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification
  • Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

Examples

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example 1

[0062]FIGS. 2A to 2F shows amplification plots generated with the CT PB1 (FIG. 2A and FIG. 2D), GC glnA7 (FIG. 2B and FIG. 2E) and GC porA7 (FIG. 2C and FIG. 2F) primers in V6.21 buffer containing V13 (FIGS. 2A, 2B and 2C) or V6.21p buffer without V13 dye (FIGS. 2D, 2E and 2F). The target sequences shown in SEQ ID NOs. 7 to 9 with CT PB1 internal probe conjugated with FAM, GC glnA7 loop probe conjugated with Joe and GC porA7 loop probe conjugated with Alexa546 respectively. All reactions were performed for 60 mins at a constant temperature of 63 C with AB17500 machine.

example 2

[0063]FIGS. 3A and 3B are melt curve analyses of LAMP products generated with CT PB1 primers in the presence of CT PB1 internal probe conjugated with FAM. 100 pg per reaction of ATTC CT DNA standard was used as a positive control. A—normalized reporter plot, B—derivative reporter plot. Melt curve plots were generated based on the readouts in FAM channel with AB17500 machine.

example 3

[0064]FIGS. 4A and B are melt curve analyses of LAMP product generated with GC glnA7 primers in the presence of GC glnA7 loop probe conjugated with JOE. 100 pg per reaction of ATTC GC DNA standard was used as a positive control. FIG. 4A shows a normalized reporter plot and FIG. 4B shows a derivative reporter plot. Melt curve plots were generated based on the readouts in JOE channel with AB17500 machine.

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Abstract

The disclosure relates to novel probes for use in LAMP detection methods. The probes contain a single fluorophore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a probe for the detection of a nucleic acid, a method using said probe and a kit of parts. Preferably the probe of the invention is useful in a method for the detection of nucleic acids derived from Chlamydia trachomatis and / or Neisseria gonorrhoeae and may be used in the diagnosis of Chlamydia and / or Gonorrhoea infections.REFERENCE TO SEQUENCE LISTING[0002]A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. §1.52(e). The name of the ASCII text file for the Sequence Listing is 23109675_1. TXT, the date of creation of the ASCII text file is Apr. 12, 2016, and the size of the ASCII text file is 17.3 KB.BACKGROUND OF THE INVENTION[0003]Nucleic acid amplification is one of the most valuable tools in the life sciences field, including application-oriented fields such as clinical medicine, in which diagnosis of infectious diseases, genetic disorders and ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6825C12Q2600/16C12Q1/6844C12Q1/689C12Q1/6816C12Q2565/107C12Q2531/119C12Q2563/107C12Q2531/101C12Q1/6888C12Q2533/101
Inventor SUWARA, MONIKA IWONAJAVED, SAJIDGILLIES, ELIZABETH ANN
Owner MAST GROUP
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