Abstract: The subject invention pertains to the detection and differentiation of genetic variations by nucleic acid amplification. The invention provides methods of detecting one or more genetic variations in a nucleic acid that are in close proximity simultaneously. The invention further provides primer and probe oligonucleotides and methods of using said primers and probes in assays to detect genetic variants of concern of SARS-CoV-2. The methods of the invention detect genetic variants of other pathogens, including influenza, or genetic variants involved in inheritable diseases or cancer.