Microarray based multiplex pathogen analysis and uses thereof

• US 10,501,814 B2
• Filed: 10/11/2018
• Issued: 12/10/2019
• 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
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US16/158,181
• Publication Number:
  US 20190085414A1
• Time in Patent Office: 425 Days
• Field of Search: None
• 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 and non-DNA nucleic acids from the pathogens harvested from the plant tissue sample;

c) amplifying, in a first amplification in a single assay, the 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 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 second fluorescent labeled bifunctional polymer linkers;

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

g) imaging the 3-dimensional lattice 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 3-dimensional lattice 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 3-dimensional lattice 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 the pathogen is a bacterium, a fungus, a virus, a yeast, an algae or a protozoan or a combination thereof.

3. The method of claim 1, wherein the pathogen is a bacterium, said first primer pair comprising the nucleotide sequences of 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.

4. The method of claim 1, wherein the pathogen is a bacterium, said first fluorescent labeled second primer pair comprising the nucleotide sequences of 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.

5. The method of claim 1, wherein the pathogen is a bacterium, said nucleic acid probes comprising the nucleotide sequences of SEQ ID NOS:

37-85.

6. The method of claim 1, wherein the pathogen is of the family Enterobacteriaceae, the first primer pair comprising the nucleotide sequences of SEQ ID NOS:

11 and 12, the first fluorescent labeled second primer pair comprising the nucleotide sequences of SEQ ID NOS;

29 and 30, and the nucleic acid probes comprising the nucleotide sequences of SEQ ID NOS;

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

7. The method of claim 1, wherein the pathogen is a fungus, said first primer pair comprising the nucleotide sequences of SEQ ID NOS:

13 and 14, or SEQ ID NOS;

15 and 16, or SEQ ID NOS;

135 and 136.

8. The method of claim 1, wherein the pathogen is a fungus, said first fluorescent labeled second primer pair comprising the nucleotide sequences of SEQ ID NOS:

31 and 32, or SEQ ID NOS;

33 and 34, or SEQ ID NOS;

139 and 140.

9. The method of claim 1, wherein the pathogen is a fungus, said nucleic acid probes comprising the nucleotide sequences of SEQ ID NOS:

86-125.

10. The method of claim 1, wherein the pathogen is Mucor spp., the first primer pair comprising the nucleotide sequences of SEQ ID NOS:

13 and 14, the first fluorescent labeled second primer pair comprising the nucleotide sequences of SEQ ID NOS;

31 and 32, and the nucleic acid probe comprising the nucleotide sequences of SEQ ID NO;

113.