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 1):
A method for quantitating copy number of DNA for one or more pathogens in a plant sample comprising the steps of:\n 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:\n a central region having a nucleotide sequence distinct from signature sequence determinants in the pathogen 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 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.

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): 0.6486644698085103
- 35 USC 102 Novelty (BERT): 0.6427132017840911
- Combined Prediction Score: 0.6480693430060684
- Mean Citation Score: 496.644404
- Max Citation Score: 717.77814
- Similarity Product: 550.6807531325627

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