Methods for detecting low levels of COVID-19 virus
Inventors
Hogan, Michael Edward • Katchman, Benjamin Alan • Eggers, Frederick Henry • Newland, Cory Scott
Assignees
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Publication Number
US-12098434-B2
Publication Date
2024-09-24
Expiration Date
Abstract
Provided herein is a method for detecting the presence of a COVID-19 virus in a human sample or an environmental sample having one or more viral and bacterial pathogens. Samples processed to obtain total nucleic acids. The nucleic acids are used as a template in a reverse transcription-amplification reaction to obtain cDNA, which is used in a PCR amplification reaction to obtain fluorescent COVID-19 virus specific amplicons. These amplicons are detected by microarray hybridization near the lowest limit of detection. Also provided is a method for detecting in addition to the COVID-19 virus, the presence of respiratory disease-causing pathogens including viruses, bacteria and fungus in a single assay using the above method.
Core Innovation
The invention relates to detecting respiratory disease-causing coronaviruses in a sample using a tandem, single-assay workflow that combines reverse transcription, PCR amplifications, microarray hybridization, and microarray imaging. The method obtains a sample, isolates total nucleic acids, and generates coronavirus specific cDNA amplicons in a first combined reverse transcription and first PCR amplification reaction, followed by fluorescent labeled virus specific amplicons in a second amplification using fluorescent labeled second primer pairs.
The fluorescent labeled coronavirus specific amplicons are hybridized to a plurality of nucleic acid probes immobilized on a microarray support. The plurality of probes comprises each of the nucleotide sequences of SEQ ID NOS: 45-66 and 85-97, and the microarray is imaged to detect fluorescent signals corresponding to the fluorescent labeled coronavirus specific amplicons.
In a COVID-19-specific embodiment, the method performs the tandem, single-assay amplification such that COVID-19 virus cDNA amplicons and at least one non-COVID-19 virus cDNA amplicon are generated from the total nucleic acid. The resulting fluorescent labeled COVID-19 virus amplicons and fluorescent labeled non-COVID-19 virus amplicons are hybridized to the same microarray probe set comprising SEQ ID NOS: 45-66 and 85-97, followed by washing and imaging to detect fluorescent signals corresponding to the COVID-19 amplicons.
Claims Coverage
The independent claims cover two microarray-based coronavirus detection methods using tandem, single-assay amplification, fluorescently labeled amplicons, and imaging. Across the claims, the microarray probes comprise nucleotide sequences of SEQ ID NOS: 45-66 and 85-97, and the COVID-19 claim includes a washing step.
Tandem single-assay combined RT and PCR to fluorescently labeled coronavirus amplicons and microarray imaging
A method for detecting at least two respiratory disease-causing coronaviruses in a sample by performing, in tandem in a single assay, a combined reverse transcription and a first PCR amplification reaction using at least two first primer pairs to generate at least two coronavirus specific cDNA amplicons, followed by a second amplification using the cDNA amplicons as template and at least two fluorescent labeled second primer pairs to generate at least two fluorescent labeled virus specific amplicons; hybridizing the fluorescent labeled coronavirus specific amplicons to a microarray of immobilized probes comprising each of the nucleotide sequences of SEQ ID NOS: 45-66 and 85-97; and imaging the microarray to detect fluorescent signals corresponding to the at least two fluorescent labeled coronavirus specific amplicons.
COVID-19 detection using combined RT and PCR with COVID-19 and non-COVID-19 amplicons, microarray hybridization, washing, and imaging
A method for detecting a COVID-19 virus in a sample by isolating total nucleic acid, performing in tandem in a single assay a combined reverse transcription and a first PCR amplification reaction using at least two first primer pairs selective for the COVID-19 virus and at least one non-COVID-19 virus to generate COVID-19 virus cDNA amplicons and non-COVID-19 virus cDNA amplicons, and performing a second amplification using the COVID-19 and non-COVID-19 cDNA amplicons as templates with at least two fluorescent labeled second primer pairs selective for a target nucleotide sequence to generate at least one fluorescent labeled COVID-19 virus amplicon and at least one fluorescent labeled non-COVID-19 virus amplicon; hybridizing the fluorescent labeled COVID-19 and non-COVID-19 virus amplicons to a microarray of immobilized probes comprising each of the nucleotide sequences of SEQ ID NOS: 45-66 and 85-97; washing the microarray at least once; and imaging the microarray to detect fluorescent signals corresponding to the at least one fluorescent labeled COVID-19 virus amplicons.
The claims center on generating virus-specific amplicons via tandem reverse transcription and PCR in a single assay, converting them to fluorescently labeled virus-specific amplicons with fluorescent labeled second primer pairs, hybridizing them to microarray probes comprising nucleotide sequences of SEQ ID NOS: 45-66 and 85-97, and imaging fluorescent signals to detect the target coronaviruses, with a washing step explicitly required in the COVID-19 claim.
Stated Advantages
Allows detection of at least two respiratory disease-causing coronaviruses in a sample.
Allows detection of COVID-19 in a sample.
Allows quantification by calculating fluorescence signal intensity where the intensity correlates with the number of virus-specific genomes or COVID-19 genomes in the sample.
Enables use of a probe plurality immobilized on a microarray support comprising nucleotide sequences of SEQ ID NOS: 45-66 and 85-97 for imaging-based detection.
Documented Applications
Detecting at least two respiratory disease-causing coronaviruses in a sample using a tandem in a single assay workflow followed by microarray hybridization and imaging.
Detecting a coronavirus 2019 (COVID-19) virus in a sample using tandem amplification including COVID-19 specific primers and at least one non-COVID-19 virus, followed by microarray hybridization, washing, and imaging.
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