Pathogenesis quantification systems and treatment methods for citrus greening blight

• US 10,351,918 B2
• Filed: 10/30/2014
• Issued: 07/16/2019
• Est. Priority Date: 10/30/2013
• Status: Active Grant

• Current Assignee: Green Life Biotech LLC
• Original Assignee: Green Life Biotech LLC
• Inventors: Lloyd, Jr., Robert M.
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/142,576
• Publication Number:
  US 20160319379A1
• Time in Patent Office: 1,720 Days
• Field of Search: None
• 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 an amount of a nucleic acid in the biological sample specific for a first organism that is a pathogen associated with a second organism, wherein in tandem the first and second organisms affect pathogenesis of a host organism, to determine a pathogen quantitative measurement,

b) quantifying an amount of a nucleic acid in the biological sample specific for the second organism, to determine a second organism quantitative measurement;

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

d) calculating a 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 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 a 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 a pathogenic index or quartile.

4. The method of claim 1, wherein said pathogen first 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 host is a citrus plant and said pathogenesis is citrus green blighting or Huanglongbin (HLB).

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

8. The method of claim 1, further comprising correlating a report algorithm or index with a pathogenic index or quartile providing clinical utility as a treatment strategy or for drug screening.

9. The method of claim 8, 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.

10. The method of claim 9, wherein multiple unique nucleic acids specific to the pathogen organisms, the host organism or the inducer are quantified and correlated in the report index.