Microarray based multiplex pathogen analysis for plants, agriculture, food, and water
Inventors
Hogan, Michael Edward • May, Melissa Rose • Eggers, Frederick Henry
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11851705-B2
Publication Date
2023-12-26
Expiration Date
Abstract
Provided is a method for a two-step multiplex DNA amplification reaction which allows bacterial or fungal DNA analysis without first extracting DNA from the sample, nor without need to enrich microbes by laboratory culture prior to analysis. Without additional DNA purification or analysis, the PCR amplified DNA is administered directly to a microarray designed to interrogate a large panel of meaningful bacteria or fungi as a single multiplex test. Microarray analysis is then performed at ambient temperature, thus enabling substantial simplification of the testing process. It is contemplated that analysis may be conducted on unprocessed and processed leaf wash and similar surface sampling of plant material, cannabis, vegetables, fruit, nuts, spices, grains, other agriculture samples, food samples, or water samples, so as to detect bacterial, yeast, mold or viral, plant or human pathogen contamination.
Core Innovation
The invention provides a method for identifying at least one bacteria or at least one fungus in a raw, unpurified sample. It performs a first PCR reaction using primer pairs selective for at least one bacterial DNA or fungal DNA to generate a first amplified product, followed by a second PCR reaction that amplifies the first amplified product using fluorescent labeled primer pairs to generate a fluorescent labeled second amplified product.
The fluorescent labeled second amplified product is applied directly to a microarray containing hybridization probes affixed thereto that are complementary to selected microbial DNA sequences. The hybridization probes consist of nucleotide sequences of SEQ ID NOS: 37-79 for bacteria and SEQ ID NOS: 80-116 for fungus, and the fluorescent product is hybridized at room temperature, washed, and optically analyzed to produce microarray binding data.
The method analyzes the microarray binding data to identify the at least one bacteria or at least one fungus in the sample. The disclosed workflow uses primer pairs for the first PCR reaction and the second PCR reaction that include at least two nucleotide sequence pairs selected from specified SEQ ID NOS for bacteria and fungi.
Claims Coverage
The claim set includes two independent claims: one directed to identifying bacteria and one directed to identifying fungi. Each independent claim defines a two-step PCR using specific selectable SEQ ID primer pairs, direct application to a microarray with SEQ ID-defined hybridization probes, room-temperature hybridization, optical analysis, and identification based on microarray binding data.
Raw, unpurified sample amplified by first and second PCR with specified primer pairs
Obtain the sample as a raw, unpurified sample; amplify DNA in the raw, unpurified sample in a first PCR reaction using primer pairs selective for at least one bacterial DNA or fungal DNA to generate a first amplified product; amplify the first amplified product in a second PCR reaction using fluorescent labeled primer pairs to generate a fluorescent labeled second amplified product; wherein the primer pairs comprise at least two nucleotide sequence pairs selected from the specified SEQ ID NOS.
Direct microarray application of fluorescent amplified product with SEQ ID-defined hybridization probes and room-temperature hybridization
Apply the fluorescent labeled second amplified product directly to a microarray containing hybridization probes affixed thereto that are complementary to selected microbial DNA sequences; wherein the hybridization probes consist of the nucleotide sequences of SEQ ID NOS: 37-79 for bacteria or SEQ ID NOS: 80-116 for fungus; hybridize the fluorescent second amplified product to the hybridization probes at room temperature and wash the microarray.
Optical microarray analysis and binding-data identification
Analyze optically hybridization of the fluorescent second amplified product to the hybridization probes to produce microarray binding data; and analyze the microarray binding data to identify the at least one bacteria or at least one fungus in the sample.
Across both independent claims, the core inventive coverage is the two-step PCR workflow performed on a raw, unpurified sample using specified SEQ ID primer pair sets, followed by direct application of the fluorescent PCR product to a microarray containing SEQ ID-defined hybridization probes. Room-temperature hybridization, optical analysis to generate microarray binding data, and identification based on that binding data complete the claimed methods for bacteria and fungi.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?