US 11,920,188 B2
Combinatorial microarray assay for clade variant detection
Frederick Henry Eggers, Sahuarita, AZ (US); Benjamin Alan Katchman, Tucson, AZ (US); Fushi Wen, Tucson, AZ (US); Candy Mavis Rivas, Tucson, AZ (US); Cory Scott Newland, Tucson, AZ (US); and Michael Edward Hogan, Stony Brook, NY (US)
Filed by Frederick Henry Eggers, Sahuarita, AZ (US); Benjamin Alan Katchman, Tucson, AZ (US); Fushi Wen, Tucson, AZ (US); Candy Mavis Rivas, Tucson, AZ (US); Cory Scott Newland, Tucson, AZ (US); and Michael Edward Hogan, Stony Brook, NY (US)
Filed on May 27, 2021, as Appl. No. 17/332,837.
Claims priority of provisional application 63/147,613, filed on Feb. 9, 2021.
Prior Publication US 2022/0251635 A1, Aug. 11, 2022
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6848 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6865 (2018.01)
CPC C12Q 1/6848 (2013.01) [C12Q 1/686 (2013.01); C12Q 1/6865 (2013.01)] 10 Claims
 
1. A method for detecting Glade variants in a Coronavirus disease 2019 virus (COVID-19) in a sample, comprising:
obtaining the sample;
harvesting viruses from the sample;
isolating a total RNA from the harvested viruses;
performing a combined reverse transcription and first amplification reaction on the total RNA using at least one first primer pair selective for all COVID-19 viruses to generate COVID-19 virus cDNA amplicons;
performing a second amplification using the COVID-19 virus cDNA amplicons as template and at least one fluorescent labeled second primer pair selective for a target nucleotide sequence in the COVID-19 virus cDNA to generate at least one fluorescent labeled COVID-19 virus amplicon;
hybridizing the fluorescent labeled COVID-19 virus amplicons to a plurality of nucleic acid probes, each having a sequence corresponding to a sequence determinant that discriminates among the Glade variants of the COVID-19 virus, said nucleic acid probes attached to a solid microarray support;
washing the microarray at least once; and
imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons, thereby detecting the Glade variants of the COVID-19 virus in the sample.