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Microarray Based Multiplex Pathogen Analysis and Uses Thereof

  • US 20190085415A1
  • Filed: 10/11/2018
  • Published: 03/21/2019
  • Est. Priority Date: 12/28/2015
  • Status: Active Grant
First Claim
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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;

    c) adding a known copy number of at least one synthetic DNA sequence as internal reference standard, 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) 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 to obtain one or more pathogen DNA-specific first amplicons and synthetic DNA-specific second amplicons;

    e) performing a second amplification in tandem using the first amplicons and the second 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 and second amplicons to obtain a pathogen DNA-specific first fluorescent labeled third amplicons and a synthetic DNA-specific first fluorescent labeled fourth amplicons respectively;

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

    g) washing the microarray at least once;

    h) imaging the microarray to detect a first fluorescent signal corresponding to the first fluorescent labeled third amplicons or the 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 with 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 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 first fluorescent signal intensity from the pathogen DNA-specific third amplicons with a first fluorescent signal intensity from the synthetic DNA-specific fourth amplicons and the known copy number of the synthetic DNA, thereby quantitating copy number of the pathogen DNA in the sample.

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