Pathogenesis Quantification Systems and Treatment Methods for Citrus Greening Blight

• US 20160319379A1
• Filed: 10/30/2014
• Published: 11/03/2016
• Est. Priority Date: 10/30/2013
• Status: Active Grant

• Current Assignee: Green Life Biotech LLC
• Original Assignee: Green Life Biotech LLC
• Inventors: Lloyd, Robert M. Jr.
• Granted Patent: US 10,351,918 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The invention relates to a novel pathogenesis model, method, or kit for detecting pathogenesis in a subject. In particular, the invention provides a pathogen index derived from a ratio of the amount of dual pathogen targets relative to the amount of host quantitative measures. The pathogen index is used in diagnosis, prognosis, and/or treatment strategy of any disease, including citrus greening diseases (HLB). Research tools and methods for screening drugs for treating or preventing citrus greening diseases, as well as treatment or prevention methods for citrus greening diseases are also provided.

Claims

1. A method for detecting pathogenesis in a biological sample, comprising:

a) quantifying the amount of a nucleic acid in the sample specific for a first organism that is a pathogen associated with a second organism that in tandem the first and second organisms affect a host organism to determine a pathogen quantitative measurement,

b) quantifying the amount of a nucleic acid in the sample specific for the host organism response to pathogen to determine a multiplex quantitation for targets associated with the pathogen of a disease; and

c) calculating the ratio of the amount of dual pathogen targets relative to the amount of host quantitative measures, wherein said ratio provides a pathogen index for detecting pathogenesis.

2. The method of claim 1, wherein said pathogen index indicates a prognosis of the pathogenesis or can be used for drug treatment efficacy and drug screening.

3. The method of claim 1, wherein the quantification of the nucleic acid in the sample specific for the first organism is performed by contacting the nucleic acid from the sample with a first set of oligonucleotide primers in a nucleic acid amplification reaction, the first set of oligonucleotides being at least partially complementary to the nucleic acid of the first organism, and wherein the quantification of the nucleic acid in the sample specific for the second organism is performed by contacting the nucleic acid from the sample with a second set of oligonucleotide primers in a nucleic acid amplification reaction, the second set of oligonucleotides being at least partially complementary to the nucleic acid of the second organism, and wherein the quantification of the nucleic acid in the sample specific for the third host organism is performed by contacting the nucleic acid from the sample with a third set of oligonucleotide primers in a nucleic acid amplification reaction, the third set of oligonucleotides being at least partially complementary to the nucleic acid of the third organism, and further comprising detecting amplicons from the nucleic acid amplification reactions, further comprising

detecting amplicons from the nucleic acid amplification reactions, wherein the amplification reactions occur in the same container, and

calculating the ratio of the amount of pathogen ratio relative to the amount of host load based on the relative number of amplicons to derive the pathogenic index or quartile.

4. The method of claim 1, wherein said pathogen organism is a virus or bacteria.

5. The method of claim 1, wherein said host organism is a bacteria, plant or animal.

6. The method of claim 1, wherein said pathogenesis is citrus green blighting or Huanglongbin (HLB).

7. The method of claim 6, wherein said host organism is Candidatus Liberibacter asiaticus (LAS).

8. The method of claim 7, wherein said pathogen organism is selected from a group consisting of a phage, virus, and any inducer or factor gene pathogenically tied together with LAS.

9. The method of claim 8, wherein said second organism is a bacteriophage.

10. The method of claim 9, wherein said bacteriophage is lytic phage selected from the group consisting of SC1, SC2, and SC3 lytic phage.

11. The method of claim 1, wherein said nucleic acid is RNA or DNA.

12. The method of claim 3, wherein said nucleic acid amplification reaction is real-time PCR, recombinase polymerase amplification (RPA), helicase-dependent amplification (HAD), loop mediate isothermal amplification (LAMP), and nicking enzyme amplification reaction (NEAR).

13. The method of claim 1, further comprising a report algorithm or index correlated with the pathogen index or quartile providing clinical utility as a treatment strategy or drug screening.

14. The method of claim 13, further comprising a step of quantifying the amount of a nucleic acid in the sample specific for an inducer of the pathogen organism to determine an inducer load;

and wherein the pathogen index and the inducer load are correlated with the report index for determining prognosis and providing clinical utility as a treatment strategy or drug screening.

15. The method of claim 14, wherein multiple unique nucleic acids specific to the pathogen organisms, the host organism or the inducer are quantified and correlated in the report index.

16. A kit for detecting pathogenesis in a sample, comprising:

a) at least a first set of oligonucleotides being at least partially complementary to a nucleic acid of a first pathogen organism that is pathogenically associated with a second organism that in tandem these two organisms affects a host organism to determine a pathogen quantitative measurement,

b) at least a second set of oligonucleotides being at least partially complementary to a nucleic acid of the host organism response to pathogen of a disease,

c) reagents for nucleic acid amplification reactions with the first and second sets of oligonucleotides;

d) instructions for conducting the nucleic acid amplification reactions and detecting one or more amplicons thereof; and

e) instructions for calculating a ratio of the amplicons or the amount of dual pathogen targets relative to the amount of host quantitative measures, wherein said ratio provides a pathogen index for detecting, prognosis, drug screening, and treatment strategy or efficacy for the pathogenesis.

17. The kit of claim 16, wherein said pathogenesis is citrus greening blight or HLB.

18. A method for screening a candidate therapeutic agent for inhibition or prevention of citrus greening blight (HLB), comprising providing a culture of Candidatus Liberibacter with a bacteriophage having a repressed phage lytic cycle;

combining the culture with a candidate therapeutic agent candidate; and

detecting inhibition of growth of the Candidatus Liberibacter culture indicating that the candidate therapeutic agent inhibits or prevents citrus greening blight (HLB).

19. The method of claim 18, wherein said is bacteriophage is a lytic phage selected from the group consisting of SC1, SC2, and SC3 lytic phage.

20. The method of claim 18, wherein the phage lytic cycle is repressed or genetically deleted.

21. A method for treating bacteriophage infection in bacteria, plant or animal individual comprising administering to the individual in need a composition comprising an effective amount of an agent selected from the group consisting of nucleoside, non-nucleoside, nucleotide, non-nucleotide, ribonucleoside, and a ribonucleoside analog that selectively inhibits viral replication.

22. A method for treating viral infection in a plant comprising administering to the plant in need a composition comprising an effective amount of an agent selected from the group consisting of nucleoside, non-nucleoside, nucleotide, non-nucleotide, ribonucleoside, and a ribonucleoside analog that selectively inhibits viral replication.

23. A method for treating citrus greening blight comprising administering to a citrus tree in need a composition comprising an effective amount of an agent selected from the group consisting of nucleoside, non-nucleoside, nucleotide, non-nucleotide, ribonucleoside, and a ribonucleoside analog that selectively inhibits viral replication.

24. The method of claim 23, wherein said composition further comprises hydroxyurea or another nucleotide pool reducer in an effective amount.

25. The method of claim 23, wherein said composition further comprises a surfactant or penetrant for enhancing delivery of said composition to a targeted location of the subject.

28. The method of claim 25, wherein said composition further comprises a surfactant or penetrant for enhancing delivery of said composition to a targeted location of the subject.

26. A method for preventing citrus greening blight comprising administering to a citrus tree in need a composition comprising an effective amount of an agent selected from the group consisting of nucleoside, non-nucleoside, nucleotide, non-nucleotide, ribonucleoside, and a ribonucleoside analog that selectively inhibits viral replication.

27. The method of claim 26, wherein said composition further comprises hydroxyurea or another nucleotide pool reducer in an effective amount.