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Microarray Based Multiplex Pathogen Analysis and Uses Thereof

  • US 20200017925A1
  • Filed: 09/24/2019
  • Published: 01/16/2020
  • Est. Priority Date: 12/28/2015
  • Status: Active Grant
First Claim
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1. A method for simultaneously quantitating copy number of DNA for one or more pathogens and a plant in a plant tissue sample, comprising the steps of:

  • a) obtaining a plant tissue sample comprising one or more pathogens;

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

    c) adding a known copy number of at least one synthetic DNA sequence as internal reference standard, each of said synthetic DNA sequences comprising;

    a central region having a nucleotide sequence distinct from signature sequence determinants in the pathogen DNA and the plant 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 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 and at least one second primer pair selective for the plant DNA to generate one or more pathogen DNA-specific first amplicons, plant DNA-specific second amplicons and synthetic DNA-specific third amplicons;

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

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

    i) superimposing the first fluorescent signal and the second fluorescent signal with the third 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 microarray with a database of signature sequence determinants for a plurality of pathogen DNAs and plant DNAs thereby identifying the pathogens and the plant in the sample;

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

    l) correlating mathematically, a second fluorescent signal intensity from the plant DNA-specific second fluorescent labeled fifth amplicons with a first fluorescent signal intensity from the synthetic DNA-specific first fluorescent labeled sixth amplicons and the known copy number of the synthetic DNA, thereby quantitating copy number of the plant DNA in the sample.

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