Combinatorial microarray assay for clade variant detection

Inventors

Eggers, Frederick Henry • Katchman, Benjamin Alan • Wen, Fushi • Rivas, Candy Mavis • Newland, Cory Scott • Hogan, Michael Edward

Assignees

PathogenDx Inc

Abstract

Provided herein is a method for detecting the presence of clade variants in the COVID-19 virus in a human sample and/or an environmental sample. Samples are processed to obtain total RNA. The RNA is used as a template in a combined reverse transcription and amplification reaction to obtain fluorescent COVID-19 virus amplicons. These amplicons are hybridized on a microarray with nucleic acid probes having sequences that discriminate among the various clade variants. The microarray is imaged to detect the clade variant and each clade variant is distinguished from others by generating an intensity distribution profile from the image, which is unique to each of the clade variants.

Core Innovation

The invention provides a combinatorial microarray assay method for detecting clade variants in a Coronavirus disease 2019 virus (COVID-19) in a sample. The method includes obtaining the sample, harvesting viruses, isolating total RNA from the harvested viruses, and performing a combined reverse transcription and asymmetric PCR amplification to generate fluorescent labeled COVID-19 virus cDNA amplicons, followed by a second amplification to generate fluorescent labeled COVID-19 virus amplicons.

The fluorescent labeled COVID-19 virus amplicons are hybridized to a plurality of nucleic acid probes attached to a solid microarray support, where each probe has a sequence corresponding to a sequence determinant that discriminates among clade variants of the COVID-19 virus. After washing and imaging the microarray to detect fluorescent signals from the hybridized amplicons, an intensity distribution profile unique to one of the clade variants is generated and used to detect the clade variant in the sample.

The described system includes clade variant look-up table support for Boolean-pattern clade identification and cloud-enabled updates for adding new clade variant probes and updating prevalent clade lists. It further describes analytical threshold values from blank RFU data converted into mutant/wild-type/universal constants for Delta calculation, together with QA/QC gating based on universal probe signal and automated reporting.

Claims Coverage

The document includes one independent claim centered on an integrated process from fluorescent asymmetric PCR through microarray hybridization, imaging, and variant calling by an intensity distribution profile unique to a clade variant. The independent claim is supported by three inventive features.

Across the independent claim, clade-variant detection is achieved by combining fluorescent labeled asymmetric PCR amplification, microarray hybridization with probes bearing clade-discriminating sequence determinants, and imaging-based generation of an intensity distribution profile unique to a clade variant.

Stated Advantages

Documented Applications