Microarray Based Multiplex Pathogen Analysis and Uses Thereof

• US 20190085414A1
• Filed: 10/11/2018
• Published: 03/21/2019
• Est. Priority Date: 12/28/2015
• Status: Active Grant

• Current Assignee: PathogenDx, Inc.
• Original Assignee: PathogenDx, Inc.
• Inventors: Hogan, Michael Edward, May, Melissa Rose, Eggers, Frederick Henry
• Granted Patent: US 10,501,814 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6893 for protozoa
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Provided herein is a dual amplification method for detecting plant pathogens by analysis of pathogen DNA in an unpurified nucleic acid sample from the plant. Pathogen-specific primers are used to generate a first set of amplicons that are further amplified in a second amplification step using fluorescent tagged pathogen-specific primers. Fluorescent amplicons thus generated are hybridized with pathogen-specific nucleic acid probes that are immobilized on a solid support using bifunctional polymer linkers. The hybridized microarray is imaged to obtain fluorescent images of the amplicons and the nucleic acid probes, which are superimposed to detect the pathogen present in the plant. Also described herein is a method to simultaneously detect both plant DNA and pathogen DNA in a single assay.

Claims

1. A method for identifying pathogens in a plant sample comprising the steps of:

a) harvesting pathogens from a plant tissue sample;

b) isolating total nucleic acids comprising DNA;

c) performing a first amplification in tandem in a single assay in the presence of plant DNA and non-DNA nucleic acids using at least one first primer pair selective for DNA isolated from the pathogens, to obtain one or more pathogen-specific first amplicons;

d) performing a second amplification in tandem using the pathogen-specific first amplicons as a template and at least one first fluorescent labeled second primer pair having a sequence complementary to an internal flanking region in the first amplicons to obtain first fluorescent labeled second amplicons;

e) hybridizing the second amplicons with nucleic acid probes specific for signature sequence determinants in the pathogen DNA, said nucleic acid probes immobilized at specific known positions on a 3-dimensional lattice microarray via a second fluorescent labeled bifunctional polymer linkers;

f) washing the microarray at least once;

g) imaging the microarray to detect a first fluorescent signal corresponding to the first fluorescent labeled second amplicons and a second fluorescent signal corresponding to the nucleic acid probes immobilized at the specific known positions on the microarray via the second fluorescent labeled bifunctional polymer linkers;

h) superimposing the first fluorescent signal with the second fluorescent signal to obtain a superimposed signal image; and

i) comparing the sequence of the nucleic acid probe at one or more superimposed signal positions on the microarray with a database of signature sequence determinants for a plurality of pathogen DNA, thereby identifying the pathogens in the sample.

2. The method of claim 1, wherein isolating total nucleic acids comprises fluidizing the plant tissue sample and centrifuging to obtain a pellet and disrupting the pellet.

3. The method of claim 1, wherein the pathogen is a human pathogen, an animal pathogen or a plant pathogen or a combination thereof.

4. The method of claim 3, wherein the pathogen is a bacterium, a fungus, a virus, a yeast, an algae or a protozoan or a combination thereof.

5. The method of claim 1, wherein the pathogen is a bacterium, said first amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

1 and 2, or SEQ ID NOS;

3 and 4, or SEQ ID NOS;

5 and 6 or SEQ ID NOS;

7 and 8, or SEQ ID NOS;

9 and 10, or SEQ ID NOS;

11 and 12, or SEQ ID NOS;

137 and 138.

6. The method of claim 1, wherein the pathogen is a bacterium, said second amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

19 and 20, or SEQ ID NOS;

21 and 22, or SEQ ID NOS;

23 and 24 or SEQ ID NOS;

25 and 26, or SEQ ID NOS;

27 and 28, or SEQ ID NOS;

29 and 30, or SEQ ID NOS;

141 and 30.

7. The method of claim 1, wherein the pathogen is a bacterium, said nucleic acid probes have sequences shown in SEQ ID NOS:

37-85.

8. The method of claim 1, wherein the pathogen is of the family Enterobacteriaceae, the first amplification forward and reverse primer pair has a sequence shown in SEQ ID NOS:

11 and 12 respectively, the second amplification forward and reverse primer pair has a sequence shown in SEQ ID NOS;

29 and 30 respectively, and the nucleic acid probes have sequences shown in SEQ ID NOS;

40-44, 50, 52, 54, 60, 62, 63, 66, 67, 73, 74, 75, 76, 77, 83.

9. The method of claim 1, wherein the pathogen is a fungus, said first amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

13 and 14, or SEQ ID NOS;

15 and 16, or SEQ ID NOS;

135 and 136.

10. The method of claim 1, wherein the pathogen is a fungus, said second amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

31 and 32, or SEQ ID NOS;

33 and 34, or SEQ ID NOS;

139 and 140.

11. The method of claim 1, wherein the pathogen is a fungus, said nucleic acid probes have sequences shown in SEQ ID NOS:

86-125.

12. The method of claim 1, wherein the pathogen is Mucor spp., the first amplification forward and reverse primer pair has a sequence shown in SEQ ID NOS:

13 and 14 respectively, the second amplification forward and reverse primer pair has a sequence shown in SEQ ID NOS;

31 and 32 respectively, and the nucleic acid probe has a sequence shown in SEQ ID NO;

113

13. A method for identifying plant DNA, comprising the steps of:

a) harvesting a plant tissue sample;

b) isolating total nucleic acids comprising DNA;

c) performing a first amplification in tandem in a single assay in the presence of pathogen DNA and non-DNA nucleic acids using at least one first primer pair selective for plant DNA, to obtain one or more plant-specific first amplicons;

d) performing a second amplification in tandem using the plant-specific first amplicons as a template and at least one first fluorescent labeled second primer pair having a sequence complementary to an internal flanking region in the first amplicons to obtain first fluorescent labeled second amplicons;

e) hybridizing the second amplicons with nucleic acid probes specific for signature sequence determinants in the plant DNA, said nucleic acid probes immobilized at specific known positions on a 3-dimensional lattice microarray via a second fluorescent labeled bifunctional polymer linkers;

f) washing the microarray at least once;

g) imaging the microarray to detect a first fluorescent signal corresponding to the first fluorescent labeled second amplicons and a second fluorescent signal corresponding to the nucleic acid probes immobilized at the specific known positions on the microarray via the second fluorescent labeled bifunctional polymer linkers;

h) superimposing the first fluorescent signal with the second fluorescent signal to obtain a superimposed signal image; and

i) comparing the sequence of the nucleic acid probe at one or more superimposed signal positions on the microarray with a database of signature sequence determinants for a plurality of plant DNA thereby identifying the plant in the sample.

14. The method of claim 13, wherein isolating total nucleic acids comprises fluidizing the plant tissue sample and centrifuging to obtain a pellet and disrupting the pellet.

15. The method of claim 13, wherein the plant is a Humulus or a Cannabis.

16. The method of claim 13, wherein the plant is Cannabis, said first amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

17 and 18, said second amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS;

35 and 36.

17. The method of claim 13, wherein the plant is Cannabis, the nucleic acid probes having sequences shown in SEQ ID NOS:

126-128.

18. A method for simultaneously identifying a pathogen and a plant in a single assay comprising the steps of:

a) harvesting a plant tissue sample comprising one or more pathogens;

b) isolating total nucleic acids comprising DNA from at least the plant tissue and the pathogens;

c) performing a first amplification in tandem in a single assay in the presence of non-DNA nucleic acids using at least one first primer pair selective for the pathogen DNA and at least one second primer pair selective for the plant DNA, to obtain pathogen-specific first amplicons and plant-specific second amplicons;

d) performing a second amplification in tandem using the first amplicons and the second amplicons as template, at least one first fluorescent labeled third primer pair having a sequence complementary to an internal flanking region in the first amplicons and at least one second fluorescent labeled fourth primer pair having a sequence complementary to an internal flanking region in the second amplicons to obtain pathogen-specific first fluorescent labeled third amplicons and plant-specific second fluorescent labeled fourth amplicons;

e) hybridizing the third amplicons and the fourth amplicons with nucleic acid probes specific for signature sequence determinants in the pathogen DNA and plant DNA respectively, said nucleic acid probes immobilized at specific known positions on a 3-dimensional lattice microarray via a third fluorescent labeled bifunctional polymer linkers;

f) washing the microarray at least once;

g) imaging the microarray to detect a first fluorescent signal corresponding to the third amplicons, a second fluorescent signal corresponding to the fourth amplicons and a third fluorescent signal corresponding to the nucleic acid probes immobilized at the specific known positions on the microarray via the third fluorescent labeled bifunctional polymer linkers;

h) superimposing each of the first fluorescent signal and the second fluorescent signal with the third fluorescent signal; and

j) comparing the sequence of the nucleic acid probe at one or more superimposed signal positions on the microarray with a database of signature sequence determinants for a plurality of pathogen DNA and plant DNA thereby identifying the pathogen and plant in the sample.

19. The method of claim 18, wherein isolating total nucleic acids comprises fluidizing the plant tissue sample and centrifuging to obtain a pellet and disrupting the pellet.

20. The method of claim 18, wherein the pathogen is a human pathogen, animal pathogen or plant pathogen or a combination thereof.

21. The method of claim 20, wherein the pathogen is a bacterium, a fungus, a virus, a yeast, an algae or a protozoan or a combination thereof.

22. The method of claim 18, wherein the pathogen is a bacterium, said first amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

1 and 2, or SEQ ID NOS;

3 and 4, or SEQ ID NOS;

5 and 6 or SEQ ID NOS;

7 and 8, or SEQ ID NOS;

9 and 10, or SEQ ID NOS;

11 and 12, or SEQ ID NOS;

137 and 138.

23. The method of claim 18, wherein the pathogen is a bacterium, said second amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

19 and 20, or SEQ ID NOS;

21 and 22, or SEQ ID NOS;

23 and 24 or SEQ ID NOS;

25 and 26, or SEQ ID NOS;

27 and 28, or SEQ ID NOS;

29 and 30, or SEQ ID NOS;

141 and 30.

24. The method of claim 18, wherein the pathogen is a bacterium, said nucleic acid probes have sequences shown in SEQ ID NOS:

37-85.

25. The method of claim 18, wherein the pathogen is a fungus, said first amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

13 and 14, or SEQ ID NOS;

15 and 16, or SEQ ID NOS;

135 and 136.

26. The method of claim 18, wherein the pathogen is a fungus, said second amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

31 and 32, or SEQ ID NOS;

33 and 34, or SEQ ID NOS;

139 and 140.

27. The method of claim 18, wherein the pathogen is a fungus, said nucleic acid probes have sequences shown in SEQ ID NOS:

86-125.

28. The method of claim 18, wherein the plant is a Humulus or a Cannabis.

29. The method of claim 18, wherein the plant is Cannabis, said first amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS:

17 and 18 and said second amplification forward and reverse primer pair having sequences shown respectively in SEQ ID NOS;

35 and 36.

30. The method of claim 18, wherein the plant is Cannabis, the nucleic acid probes having sequences shown in SEQ ID NOS:

126-128.