PATENT CLAIM ANALYSIS

Application Number: 16158276
Application Type: Utility
Filing Date: 2018-10
Publication Date: 2019-03
Patent Classification: ["435", "006100"]

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.

Claim (Index 16):
A method for simultaneously quantitating copy number of DNA for one or more pathogens and plants in a plant tissue sample in single assay comprising the steps of:\n a) harvesting the plant tissue sample comprising one or more pathogens; b) isolating total nucleic acids comprising at least the plant tissue DNA and the pathogen 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:\n a central region having a nucleotide sequence distinct from signature sequence determinants in the pathogen DNA and the plant DNA; and \n a 5\u2032 end and a 3\u2032 end having nucleotide sequences substantially identical to a consensus sequence in the pathogen DNA; \n d) performing a first amplification in tandem in a single assay in the presence of non-DNA 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 obtain pathogen DNA-specific first amplicons, plant DNA-specific second amplicons and synthetic DNA-specific third amplicons; e) performing a second amplification in tandem using the first amplicons, the second amplicons and the third amplicons as a template, at least one first fluorescent labeled third primer pair having a sequence complementary to an internal flanking region in the first amplicons and third amplicons and, at least one second fluorescent labeled fourth primer pair having a sequence complementary to an internal flanking region in the second amplicons to obtain a 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 fourth, fifth and sixth amplicons with nucleic acid probes specific for signature sequence determinants in the pathogen DNA, plant DNA and synthetic DNA respectively, said nucleic acid probes immobilized at specific known positions on a 3-dimensional lattice microarray via a third 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 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 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 DNA and plant DNA thereby identifying the pathogens and plant in the sample; k) correlating mathematically, a first fluorescent signal intensity from the pathogen DNA-specific fourth amplicons with a first fluorescent signal intensity from the synthetic DNA-specific 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 fifth amplicons with a first fluorescent signal intensity from the synthetic DNA-specific sixth amplicons and the known copy number of the synthetic DNA, thereby quantitating copy number of the plant DNA in the sample.

Metadata:
- Claim Count in Document: 52.0
- Percentile: 97.0
- Lexical Diversity: 2.08955
- Patent Class: 435.0
- Transitional Phrase Type: open
- Component Type: 1
- Foreign Priority: False
- Related Applications: ['16158181', '15916036', '12100519', '09865807', '14694008']

Analysis Scores:
- 35 USC 101 Eligibility (BERT): 0.653299294642109
- 35 USC 102 Novelty (BERT): 0.6296393404598448
- Combined Prediction Score: 0.6509332992238825
- Mean Citation Score: 496.644404
- Max Citation Score: 717.77814
- Similarity Product: 512.9558089477443

Labels:
- Claim Label 101: 1
- Claim Label 102: 1
- Claim Label 103: 1
- Claim Label 112: 1
- Combined Label: 1
- Label 101 Adjusted: 1

Dataset: test