Microarray based multiplex pathogen analysis and uses thereof

• US 10,612,075 B2
• Filed: 03/08/2018
• Issued: 04/07/2020
• 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
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US15/916,036
• Publication Number:
  US 20180251822A1
• Time in Patent Office: 761 Days
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide

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 detecting pathogens in a plant sample, comprising the steps of:

a) harvesting pathogens from a Cannabis tissue sample;

b) isolating total nucleic acids comprising DNA and non-DNA nucleic acids from the pathogens harvested from the Cannabis tissue sample;

c) amplifying, in a first amplification in a single assay pathogen DNA in the total nucleic acids using at least one first primer pair selective for at least one pathogen-specific DNA isolated from the harvested pathogens to generate one or more pathogen-specific first amplicons;

d) amplifying, in a second amplification using the one or more pathogen-specific first amplicons as a template and at least one first fluorescent labeled second primer pair to generate first fluorescent labeled second amplicons;

e) hybridizing the first fluorescent labeled second amplicons to a plurality of nucleic acid probes each having a sequence corresponding to sequence determinants in a plurality of pathogen DNAs, said nucleic acid probes attached covalently to a plurality of second fluorescent labeled bifunctional polymer linkers at specific known positions on a 3-dimensional lattice microarray;

f) washing the 3-dimensional lattice microarray at least once; and

g) imaging the 3-dimensional lattice microarray to detect a first fluorescent signal from the first fluorescent labeled second amplicons thereby detecting the pathogens in the plant sample.

2. The method of claim 1, further comprising the steps of imaging the 3-dimensional lattice microarray to detect a second fluorescent signal from the nucleic acid probes at the specific known positions on the 3-dimensional lattice microarray and superimposing the first fluorescent signal image on the second fluorescent signal image to produce a superimposed signal image, thereby identifying the pathogens detected in the plant sample.

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, an yeast, an algae or a protozoan or a combination thereof.

5. The method of claim 4, wherein the pathogen is a bacterium, said first primer pair comprising the nucleotide sequences of SEQ ID NOS:

1-12.

6. The method of claim 4, wherein the pathogen is a bacterium, said first fluorescent labeled second primer pair comprising the nucleotide sequences of SEQ ID NOS:

19-30.

7. The method of claim 4, wherein the pathogen is a bacterium, said nucleic acid probes comprising the nucleotide sequences of SEQ ID NOS:

37-85.

8. The method of claim 4, wherein the pathogen is a fungus, said first primer pair comprising the nucleotide sequences of SEQ ID NOS:

13-16.

9. The method of claim 4, wherein the pathogen is a fungus, said first fluorescent labeled second primer pair comprising the nucleotide sequences of SEQ ID NOS:

31-34.

10. The method of claim 4, wherein the pathogen is a fungus, said nucleic acid probes comprising the nucleotide sequences of SEQ ID NOS:

86-125.