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BR-112019001623-B1 - Ligand Binding Assay Kit for Detecting the Presence of Gibberellins in a Sample

BR112019001623B1BR 112019001623 B1BR112019001623 B1BR 112019001623B1BR-112019001623-B1

Abstract

The invention relates to the detection of gibberellins and, more specifically, but not exclusively, to the detection of gibberellins in barley. A problem in the art is that the test to detect gibberellins requires special equipment, making it expensive and time-consuming. It is, therefore, an object of this invention to provide a ligand-binding assay for detecting gibberellins, in which the above disadvantages can, at least partially, be overcome or mitigated and/or provide a more useful alternative to the present art to a greater extent. It is contemplated that the invention will provide a point of use of a ligand-binding assay for detecting gibberellins in barley seeds through the use of aptamers.

Inventors

  • CHARLES STEPHEN WHITEHEAD
  • Eduard VENTER
  • JOSEPH ADRIAN WALKER
  • KHA QUAN TRAM

Assignees

  • UNIVERSITY OF JOHANNESBURG

Dates

Publication Date
20260310
Application Date
20170726
Priority Date
20160726

Claims (13)

  1. 1. Ligand binding assay kit for detecting the presence of gibberellins in a sample, characterized in that it comprises: - a target binding element selected to bind to gibberellin selected from the group consisting of GA1, GA3, GA4 and GA7; wherein the target binding element is associated with a visual indicator that indicates the presence of gibberellin in the sample when the target binding element binds to gibberellin, wherein the target binding element is an aptamer.
  2. 2. Assay kit according to claim 1, characterized in that the aptamer has 62 nucleotides with the nucleic acid sequence: 5’-NHMCVNNNHDGCTGAGGTATGCNNNHWNDYDDNDNNHHHNHNVVHNNNNNDNNDBNHNNNHD-3’(SEQ ID NO: 12), - wherein Y is T or C; - wherein M is A or C; - wherein W is A or T; - wherein B is G, C or T; - wherein D is A, G or T; - wherein H is A, C or T; - wherein V is A, G or C; - wherein N is A, G, C or T; or its complement, or an RNA equivalent of the molecule or its complement.
  3. 3. Assay kit according to claim 1, characterized in that the aptamer has 62 nucleotides with the nucleic acid sequence: 5’-TGAGGDVNVNGCTGAGGTATGCMAAYDHMHNVNNNNNHNNNNNNNNNNNNNNNNNNNDHNNN-3’ (SEQ ID NO: 13), - wherein Y is T or C; - wherein M is A or C; - wherein D is A, G or T; - wherein H is A, C or T; - wherein V is A, G or C; - wherein N is A, G, C or T; or its complement, or an RNA equivalent of the molecule or its complement.
  4. 4. Assay kit according to claim 1, characterized in that the aptamer consists of a nucleic acid sequence of sequences SEQ ID NO: 1 to 11.
  5. 5. Test kit according to claim 1, characterized in that the ligand binding test is a lateral or vertical flow, in which aptamers are bound to a carrier and the indicator is provided in the form of a binding signal of the saturation of the aptamer binding sites on the carrier.
  6. 6. Assay kit according to claim 5, characterized in that the assay is an inhibition assay.
  7. 7. Test kit according to claim 5, characterized in that the carrier is a gold nanoparticle bonded to the aptamer by thiolation.
  8. 8. Assay kit according to claim 7, characterized in that the surface plasmonic resonance of a highly localized concentration of gold nanoparticles provides a visual indicator in the absence of gibberellins in the sample.
  9. 9. Test kit according to claim 8, characterized in that the visual indicator is perceptible as a visible red signal in a portion of the lateral or vertical flow test indication.
  10. 10. Test kit according to claim 9, characterized in that the visible red signal is a result of the saturating binding sites of the gold nanoparticle complex attached to the aptamer in the carrier, thus preventing the aggregation of the gold particles.
  11. 11. Assay kit according to claim 1, characterized in that the assay is a non-radioactive ligand binding assay that includes aptamers that are chemically linked to a fluorophore and quencher pair such that the visual indicator is provided in the form of fluorescence in the presence of gibberellins.
  12. 12. Assay kit according to claim 11, characterized in that the fluorophore and suppressor pair is a nucleic acid dye and a fluorescent hybridization probe.
  13. 13. Assay kit according to claim 1, characterized in that the ligand binding assay includes aptamers that are chemically linked to an acceptor-donor pair such that the visual indicator is provided in the form of fluorescence in the presence of gibberellins.

Description

FIELD OF THE INVENTION [0001] The invention relates to a ligand binding assay and more specifically, but not exclusively, to a ligand binding assay for the detection of gibberellins in barley. BACKGROUND OF THE INVENTION [0002] Gibberellins or gibberellic acids (GAs) are plant hormones that regulate growth and influence various developmental processes, including stem elongation, germination, dormancy, flowering, sexual expression, enzyme induction, and senescence of leaves and fruits. [0003] All known gibberellins are tetracyclic diterpenic acids that are synthesized via the terpenoid pathway in plastids and then modified in the endoplasmic reticulum and cytosol until they reach their biologically active form. All gibberellins are derived via the ent-gibberellane skeleton, but are synthesized via ent-kaurene. [0004] One problem with the technique is that the test to detect gibberellins requires specialized equipment, making it expensive and time-consuming. OBJECT OF THE INVENTION [0005] It is, therefore, an objective of this invention to provide a ligand binding assay for detecting gibberellins that, at least partially, mitigates the problem associated with the prior art. SUMMARY OF THE INVENTION [0006] According to a first aspect of the invention, a ligand binding assay is provided for detecting the presence of gibberellins in a sample comprising: - a target binding element selected to bind to gibberellins; and - the target binding element being associated with a visual indicator, such that the visual indicator indicates the presence of gibberellins in the sample when the target binding element binds to gibberellins. [0007] Gibberellins are the gibberellic acids GA1, GA3, GA4 or GA7. [0008] The linking element to the target is a target portion. The portion is an aptamer. [0009] The aptamer has 62 nucleotides with the nucleic acid sequence 5’-NHMCVNNNHDGCTGAGGTATGCNNNHWNDYDDNDNNHHHNHNVVHNNNNNDN NDBNHNNNHD- 3’ (SEQ ID NO: 12), wherein Y is T or C; wherein M is A or C; wherein W is A or T; wherein B is G or C or T; wherein D is A or G or T; wherein H is A or C or T; wherein V is A or G or C; wherein N is A or G or C or T, unknown or other; or its complement, or an RNA equivalent of the molecule or its complement. [00010] The aptamer has 62 nucleotides with the nucleic acid sequence 5’-TGAGGDVNVNGCTGAGGTATGCMAAYDHMHNVNNNNNHNNNNNNNNNNNNNNNN NNNNDHNNN - 3' (SEQ ID NO: 13) wherein Y is T or C; wherein M is A or C; wherein D is A or G or T; wherein H is A or C or T; wherein V is A or G or C; wherein N is A or G or C or T, unknown or other; or its complement, or an RNA equivalent of the molecule or its complement. [00011] The aptamer may comprise a nucleic acid sequence having at least 90% homology to the sequence selected from a G1-G10 group consisting of the following nucleic acid sequences selected from a group consisting of SEQ ID NO: 1-11, or its complement, or an RNA equivalent of the molecule or its complement. [00012] The binder binding test can be a lateral or vertical flow test, in which aptamers are bound to a carrier and the indicator is provided in the form of a binding signal from the saturation of the aptamer binding sites on the carrier. [00013] The lateral or vertical flow assay can be an inhibition assay. [00014] The carrier can be gold nanoparticles and the gold nanoparticles can be linked to the aptamer by thiolation. [00015] Surface plasmonic resonance of a highly localized concentration of gold nanoparticles results in a visual indicator in the absence of gibberellins in the sample. The visual indicator may be perceptible as a visible red signal in a portion of the lateral or vertical flow assay indication. [00016] Depletion of the binding signal from saturation of the aptamer binding sites on gold nanoparticles results in the absence of the red signal in the presence of gibberellins in the sample. [00017] The visible red signal is a result of the gold nanoparticle complex bound to the aptamer saturating the binding sites on the carrier and thus preventing the aggregation of the gold particles. [00018] The binder binding test may be in the form of a disposable lateral flow or vertical flow test with the product applied to a disposable test membrane. [00019] The disposable vertical flow test can be a standard “sandwich” type vertical flow test or a competitive vertical flow test. [00020] The ligand binding assay can be a non-radioactive ligand binding assay that includes aptamers that are chemically linked to a fluorophore and quencher pair such that the visual indicator is provided in the form of fluorescence in the presence of gibberellins. [00021] The fluorophore and suppressor pair can be a nucleic acid dye and a fluorescent hybridization probe. [00022] The ligand binding assay includes aptamers that are chemically linked to an acceptor-donor pair such that the visual indicator is provided in the form of fluorescence in the presence of gibberellins. BRIEF DESCRIPTION OF THE ATTACHED DIAGRAM