Microarray based multiplex pathogen analysis and uses thereof

• US 10,513,745 B2
• Filed: 10/11/2018
• Issued: 12/24/2019
• 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:
  US16/158,276
• Publication Number:
  US 20190085415A1
• Time in Patent Office: 439 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• 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 an internal standard method for determining copy number of a pathogen DNA in an unpurified nucleic acid sample by using a known copy number of synthetic DNA that shares a consensus region sequence with the pathogen. The sample is subject to two amplification steps using locus-specific primers and fluorescent primers respectively to obtain fluorescent amplicons for the pathogen and synthetic DNA. These are hybridized with immobilized pathogen-specific and synthetic DNA-specific nucleic acid probes and imaged to obtain fluorescent signals for pathogen-specific and synthetic DNA-specific amplicons. Signal intensities are correlated with the known copy number of synthetic DNA to determine copy number of pathogen DNA in the plant. Also described herein is a method to simultaneously quantitate using the above method, copy numbers of both pathogen and plant DNA in a sample.

Claims

1. A method for quantitating copy number of DNA for one or more 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) adding a known copy number of at least one synthetic DNA sequence as an internal reference standard to the total nucleic acids, each of said synthetic DNA sequence comprising;

a central region having a nucleotide sequence distinct from signature sequence determinants in the pathogen DNA; and

a 5′

end and a 3′

end having nucleotide sequences substantially identical to a consensus sequence in the pathogen DNA;

d) amplifying, in a first amplification in tandem in a single assay the pathogen DNA and the at least one synthetic DNA sequence in the total nucleic acids using at least one first primer pair selective for the pathogen DNA and the synthetic DNA sequence to generate one or more pathogen DNA-specific first amplicons and one or more synthetic DNA-specific second amplicons;

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

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

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

h) imaging the 3-dimensional lattice microarray to detect a first fluorescent signal corresponding to the pathogen DNA-specific first fluorescent labeled third amplicons and the synthetic DNA-specific first fluorescent labeled fourth 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;

i) superimposing the first fluorescent signal on the second fluorescent signal to obtain a superimposed signal image;

j) 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; and

k) correlating mathematically, a fluorescent signal intensity from the pathogen DNA-specific first fluorescent labeled third amplicons with a fluorescent signal intensity from the synthetic DNA-specific first fluorescent labeled fourth amplicons and the known copy number of the synthetic DNA sequence, thereby quantitating copy number of the pathogen DNA in the sample.

2. The method of claim 1, wherein the synthetic DNA comprises one of the nucleotide sequences of SEQ ID NOS:

154-157.

3. 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.

4. The method of claim 1, wherein the pathogen is a bacterium, said consensus sequence comprising the nucleotide sequence of SEQ ID NO:

153, the 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.

5. The method of claim 1, wherein the pathogen is a bacterium, said consensus sequence comprises the nucleotide sequence of SEQ ID NO:

153, the first fluorescent 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.

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

37-85.

7. The method of claim 1, wherein the pathogen is a bacterium, the synthetic DNA comprising the nucleotide sequences of SEQ ID NO:

155, SEQ ID NO;

156 and SEQ ID NO;

157 that hybridize to synthetic DNA specific nucleic acid probes comprising the nucleotide sequences of SEQ ID NO;

142, SEQ ID NO;

143 and SEQ ID NO;

144, respectively.

8. The method of claim 1, wherein the pathogen is a fungus, said consensus sequence comprising the nucleotide sequence of SEQ ID NO:

152, the 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.

9. The method of claim 1, wherein the pathogen is a fungus, said consensus sequence comprising the nucleotide sequence of SEQ ID NO:

152, the 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.

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

86-125.

11. The method of claim 1, wherein the pathogen is a fungus, the synthetic DNA comprising the nucleotide sequence of SEQ ID NO:

154 that hybridizes to synthetic DNA specific nucleic acid probes comprising the nucleotide sequence of SEQ ID NO;

145.

12. The method of claim 1, wherein the pathogen is a fungus, the first primer pair comprising the nucleotide sequences of SEQ ID NO:

135 and 136, the first fluorescent labeled second primer pair comprising the nucleotide sequences of SEQ ID NO;

139 and 140, the synthetic DNA sequence comprising the nucleotide sequence of SEQ ID NO;

154, the nucleic acid probe comprising the nucleotide sequence of SEQ ID NO;

86, and the synthetic DNA specific nucleic acid probe comprising the nucleotide sequence of SEQ ID NO;

145.

13. The method of claim 1, wherein the step of correlating mathematically is performed using the relationship:

C_n/C_o=P(S_n/S_o)^x where,

C_n is the copy number of pathogen DNA of each type n of pathogen species to be quantitated, C_o is the known copy number of the synthetic DNA sequence added to the total nucleic acids prior to the performing a first amplification step, S_n is the first fluorescent signal intensity for each of the type n of pathogen species, in relative fluorescence units (RFU) from the pathogen DNA-specific first fluorescent labeled third amplicons that hybridized to the pathogen DNA-specific nucleic acid probes at the specific known positions on the microarray, S_o is fluorescent signal intensity in RFU from the synthetic DNA-specific first fluorescent labeled fourth amplicons that hybridized to the synthetic DNA-specific nucleic acid probes at the specific known positions on the 3-dimensional lattice microarray, P is a constant ranging from about 0.1 to about 10 and X is a an exponential factor ranging from about 1 to about 3.