Compositions and methods for detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Influenza A and Influenza B
Inventors
Manohar, Chitra • Fontecha, Marcel R. • Heil, Marintha • Ravirala, Ramani Sadanandam • Santini, Christopher David • Spier, Eugene • Sun, Jingtao • Tam, Thanh • Truong, Huan • Yee, Michelle Elizabeth • Mangipudi, Kalyani
Assignees
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Publication Number
US-12195811-B2
Publication Date
2025-01-14
Expiration Date
Abstract
Methods for the rapid detection of the presence or absence of SARS-CoV-2, influenza A and influenza B in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting SARS-CoV-2, influenza A, and influenza B and kits are provided that are designed for the detection of SARS-CoV-2, influenza A and influenza B.
Core Innovation
The invention provides viral diagnostic methods and kits for rapid qualitative/quantitative RT-PCR detection of SARS-CoV-2. The workflow includes reverse transcription, amplification and hybridization/detection, and detection is based on contacting amplification products with detectable probes and detecting the presence or absence of the amplification products to indicate the presence or absence of SARS-CoV-2 in a sample.
The invention further enables multiplex detection by simultaneously detecting SARS-CoV-2, influenza A, and influenza B in a single sample using three distinct primer sets and three or more detectable probes. Each primer set produces an amplification product if its corresponding target nucleic acid is present, and at least one probe is specific for the amplification products of each primer set, enabling detecting the presence or absence of amplified products indicative of the presence or absence of SARS-CoV-2, influenza A, and influenza B.
The disclosed approach also includes an additional primer set to produce a further amplification product for SARS-CoV-2, including SARS-CoV-2 and other coronavirus target nucleic acids from the subgenus Sarbecovirus, with a corresponding detectable probe. The invention specifies particular primer and probe oligonucleotide sequences using SEQ ID NOs, including first and second primer/probe sets for SARS-CoV-2, a second set for influenza A, and a third set for influenza B.
Claims Coverage
The document includes two independent claims: one directed to a method for simultaneously detecting SARS-CoV-2, influenza A, and influenza B, and one directed to a kit for the same multiplex detection. Each independent claim is characterized by three primer sets producing respective amplification products and three or more detectable probes that specifically hybridize to those amplification products, with sequence-defined primer and probe oligonucleotide selections using SEQ ID NOs.
Simultaneous amplification with three primer sets for SARS-COV-2, influenza A, and influenza B
A method in which a sample is contacted with a first set of primers, a second set of primers, and a third set of primers to produce amplification products if SARS-COV-2, influenza A, and/or influenza B is present, where the first set produces an amplification product if SARS-COV-2 is present, the second set produces an amplification product if influenza A is present, and the third set produces an amplification product if influenza B is present, with the primer oligonucleotide sequences selected from specified SEQ ID NO groups for each primer set.
Hybridizing amplification products with probes specific to each target amplification product
A hybridizing step in which the amplification product(s) are contacted with three or more detectable probes, wherein the detectable probes include at least one probe specific for the amplification products of each of the first, second, and third sets of primers, with the first, second, and third detectable probes selected from specified SEQ ID NO groups for each corresponding target amplification product.
Detecting presence or absence of amplified products to indicate target presence
Detecting the presence or absence of the amplified products, where the presence of an amplification product is indicative of the presence of SARS-COV-2, and/or influenza A, and/or influenza B in the sample, and the absence of an amplification product is indicative of the absence of SARS-COV-2, and/or influenza A, and/or influenza B in the sample.
Sequence-defined primer/probe multiplex kit for SARS-COV-2, influenza A, and influenza B
A kit that simultaneously detects one or more nucleic acids of SARS-COV-2, one or more nucleic acids of influenza A, and one or more nucleic acids of influenza B in a sample, comprising a first set of primers that produces a first amplification product if SARS-COV-2 is present, a second set of primers that produces a second amplification product if influenza A is present, and a third set of primers that produces a third amplification product if influenza B is present, and three or more detectable probes comprising a first detectable probe that specifically hybridizes to the first amplification product, a second detectable probe that specifically hybridizes to the second amplification product, and a third detectable probe that specifically hybridizes to the third amplification product, with each primer and probe comprising or consisting of oligonucleotide sequences selected from specified SEQ ID NO groups.
Across the independent claims, the coverage centers on multiplex detection of SARS-COV-2, influenza A, and influenza B using three primer sets that generate corresponding amplification products, three or more detectable probes that specifically hybridize to each target amplification product, and detecting presence or absence of amplified products to indicate target presence or absence, with the kit and assay components defined by specific SEQ ID NO selections.
Stated Advantages
Rapid qualitative/quantitative RT-PCR detection of SARS-CoV-2.
Simultaneous multiplex detection of SARS-CoV-2, influenza A, and influenza B in a sample.
Documented Applications
Diagnosing or detecting SARS-CoV-2 in a sample using RT-PCR, including qualitative/quantitative detection.
Simultaneous detection of SARS-CoV-2, influenza A, and influenza B in a single sample using multiplex RT-PCR.
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