Methods, reagents and kits for detection of Karnal bunt

• US 20200131590A1
• Filed: 10/29/2019
• Published: 04/30/2020
• Est. Priority Date: 10/29/2018
• Status: Active Application

• Current Assignee: New South Wales Department of Industry
• Original Assignee: New South Wales Department of Industry
• Inventors: Tan, Mui-Keng
• Application Number:
  US16/666,675
• Publication Number:
  US 20200131590A1
• Time in Patent Office: Days
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A01N 25/00 Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application , e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: G01N 2333/37 from fungi
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/56961 Plant cells or fungi

Abstract

The present disclosure relates to methods and test kits for detecting Tilletia indica (Karnal bunt) in biological samples. In particular, the present disclosure relates to a diagnostic method employing isothermal amplification of nucleic acids, namely a loop-mediated amplification (LAMP), in combination with lateral flow technology to detect Karnal bunt in a grain sample, e.g., from wheat or triticale, as well as test kits and reagent mixtures for use in same.

Claims

1. A test kit for detecting Tilletia indica or spores thereof in a sample, said test kit comprising:

(1) a reagent mixture comprising reagents configured to amplify a nucleic acid sequence of Tilletia indica by an isothermal amplification assay to produce a biotinylated amplification product comprising a sequence set forth in SEQ ID NO;

1;

(2) a nucleic acid probe comprising a polynucleotide sequence of at least 16 nucleotides in length which is sufficiently complementary to a region of corresponding length within the biotinylated amplification product such that the nucleic acid probe and amplification product are hybridisable, wherein the nucleic acid probe is conjugated to a hapten;

(3) one or more lateral flow test strips comprising

(a) a label-holding portion comprising a mobilisable capture reagent comprising a detectable label, wherein the mobilisable capture reagent is configured to bind to the hapten conjugated to the nucleic acid probe; and

(b) a test portion comprising an immobilised capture reagent configured to specifically bind biotin and thereby immobilise biotin to the test portion.

2. The test kit according to claim 1, wherein the reagent mixture comprises:

at least one DNA polymerase enzyme;

isothermal amplification primers specific for Tilletia indica;

dinucleotide triphosphates (dNTPs);

one or more salts; and

a buffer.

3. The test kit according to claim 1, wherein the isothermal amplification assay is selected from loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and helicase-dependent amplification (HDA).

4. The test kit according to claim 1, wherein the isothermal amplification assay is a LAMP assay.

5. The test kit according to claim 4, wherein the reagent mixture comprises:

at least one DNA polymerase enzyme;

LAMP primers specific for Tilletia indica;

dinucleotide triphosphates (dNTPs);

a magnesium salt; and

a buffer.

6. The test kit according to claim 5, wherein the reagent mixture comprises the following LAMP primers:

Ti-FIP comprising the sequence set forth in SEQ ID NO;

2 or a sequence which is substantially identical thereto, wherein the sequence of Ti-FIP is conjugated to a biotin;

Ti-BIP comprising the sequence set forth in SEQ ID NO;

3 or a sequence which is substantially identical thereto;

Ti-LF comprising the sequence set forth in SEQ ID NO;

4 or a sequence which is substantially identical thereto;

Ti-LB comprising the sequence set forth in SEQ ID NO;

5 or a sequence which is substantially identical thereto;

Ti-F3 comprising the sequence set forth in SEQ ID NO;

6 or a sequence which is substantially identical thereto; and

Ti-B3 comprising the sequence set forth in SEQ ID NO;

7 or a sequence which is substantially identical thereto.

7. The test kit according to claim 6, wherein the LAMP primers are:

Ti-FIP (SEQ ID NO;

2) conjugated to a biotin;

Ti-BIP (SEQ ID NO;

3);

Ti-LF (SEQ ID NO;

     4);

Ti-LB (SEQ ID NO;

     5);

Ti-F3 (SEQ ID NO;

     6); and

Ti-B3 (SEQ ID NO;

     7).

8. The test kit according to claim 5, wherein

(i) the DNA polymerase enzyme is selected from the groups consisting of Bst DNA polymerase, Bsm DNA polymerase, Gst DNA polymerase, SD DNA polymerase and combinations thereof;

and/or

(ii) the magnesium salt is MgSO₄.

9. The test kit according to claim 1, wherein:

(i) the reagent mixture is thermostable; and

/or

(ii) the reagent mixture and the nucleic acid probe are each provided in a dried form.

10. The test kit according to claim 9, wherein the reagent mixture comprises a cryoprotectant.

11. The test kit according to claim 10, wherein:

(i) the cryoprotectant is sucrose or trehalose; and

/or

(ii) the cryoprotectant is present at about 7% w/v to about 8% w/v.

12. The test kit according to claim 1, wherein the reagent mixture is provided in a liquid or frozen form and the nucleic acid probe is provided in a dried form.

13. The test kit according to claim 1, wherein the nucleic acid probe is substantially identical to the polynucleotide sequence set forth in SEQ ID NO:

8 or comprises the polynucleotide sequence set forth in SEQ ID NO;

8.

14. The test kit according to claim 1, wherein:

(a) the reagent mixture is thermostable and comprises;

(i) a Bst DNA polymerase

(ii) the LAMP primers;

Ti-FIP (SEQ ID NO;

     2) conjugated to a biotin;

Ti-BIP (SEQ ID NO;

     3);

Ti-LF (SEQ ID NO;

     4);

Ti-LB (SEQ ID NO;

     5);

Ti-F3 (SEQ ID NO;

     6); and

Ti-B3 (SEQ ID NO;

     7);

(iii) dNTPs;

(iv) MgSO₄; and

(v) a buffer; and

(b) the nucleic acid probe comprises the polynucleotide sequence set forth in SEQ ID NO;

8.

15. The test kit according to claim 1, wherein:

(i) the immobilised capture reagent is a biotin ligand;

(ii) the mobilisable capture reagent of the label-holding portion is an antibody which binds the hapten, and wherein the antibody is conjugated to the detectable label;

(iii) the hapten is a fluorophore selected from FITC or FAM, or digoxigenin;

(iv) the detectable label is a gold nanoparticle, latex nanoparticle or a fluorescent quantum dot (QD);

(iv) the one or more test strips comprise a control portion comprising an immobilised capture reagent configured to detect the mobilisable capture reagent

(v) the one or more test strips comprises a sample receiving portion for receiving a liquid sample.

16. The test kit according to claim 15, wherein:

(i) the immobilised capture reagent at the control portion is an antibody against the mobilisable capture reagent; and

/or

(ii) the control portion is located downstream of the test portion on the test strip.

17. The test kit according to claim 1, comprising:

(i) an aqueous buffer for the test strip(s);

(ii) an assay positive control comprising isolated Tilletia indica DNA or a fragment thereof suitable as a template for the isothermal amplification assay;

(iii) an apparatus configured to receive the test strip(s) and present information about the identification of Tilletia indica or spores thereof in a sample to a user via a display.

18. The test kit according to claim 17, wherein:

(i) the apparatus is provided in the form of a hand-held device; and

/or

(ii) the apparatus comprises a reader to identify Tilletia indica or spores thereof in the sample.

19. The test kit according to claim 18, wherein the reader includes one or more photodetectors capable of monitoring light reflection or light output at the test portion.

20. A method of detecting the presence or absence of Tilletia indica nucleic acids in a sample, said method comprising:

(a) performing an isothermal amplification assay on DNA extracted from the sample (sample DNA) using a reagent mixture of the test kit according to claim 1;

(b) incubating the product of (a) in the presence of a nucleic acid probe of the test kit according to claim 1;

(c) contacting the product of (b) with the sample receiving portion of a test strip of the test kit according to claim 1;

(d) detecting the presence or absence of an amplification product at the test portion of the test strip; and

(e) determining the presence or absence of Tilletia indica nucleic acids in the sample based on the presence or absence of an amplification product at the test portion.

21. The method according to claim 20, wherein:

(i) detecting a signal at the test portion is indicative of the presence of Tilletia indica nucleic acids in the sample; and

(ii) detecting no signal at the test portion is indicative of the absence of Tilletia indica nucleic acids in the sample.

22. The method according to claim 20, wherein one or more of the following applies:

(i) the detecting a signal at the control portion of the test strip is indicative that the sample has flowed through the test strip;

(ii) a volume of buffer is added to the sample following the incubation at (b) and prior to contacting the sample with the sample receiving portion of the test strip;

(iii) the reagent mixture is provided in a dried form and water is added to thermostable reagent mixture at (a);

(iv) the isothermal amplification assay is performed at about 60°

C. to about 66°

C.;

(v) the isothermal amplification assay is a loop-mediated isothermal amplification assay (LAMP);

(vi) the incubation step at (b) is performed at about 64°

C.; and

/or

(vii) the sample is grain or a part thereof, a commodity comprising grain or a part thereof, or debris comprising grain or a part thereof as a contaminant.

23. The method according to claim 22, wherein the grain is from wheat or triticale.