Polynucleotides for amplification and detection of SARS-CoV-2
Inventors
Andini, Nadya • Chiu, Kathy • Jiang, Xuewen • Maamar, Hédia
Assignees
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Publication Number
US-12264365-B2
Publication Date
2025-04-01
Expiration Date
Abstract
Disclosed herein are primers and probes related to the detection of SARS-CoV-2 via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of SARS-CoV-2 in test samples and/or to diagnose Covid-19. Specifically, the present disclosure describes primers and probes that bind to the N gene, ORF1ab, or E gene of SARS-CoV-2 coronavirus for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.
Core Innovation
The document describes a SARS-CoV-2 NAAT based on loop-mediated isothermal amplification, using RTLAMP with a strand displacement DNA polymerase and reverse transcriptase. Detection targets multiple genes including ORF1ab, N, and E, using sequence-specific primer sets such as Set-1 through Set-17.
In particular, the document specifies a primer set comprising forward outer primer SEQ ID NO: 63, backward outer primer SEQ ID NO: 86, forward inner primer SEQ ID NO: 65, backward inner primer SEQ ID NO: 87, forward loop primer SEQ ID NO: 67, and backward loop primer SEQ ID NO: 88, including primer components corresponding to outer, inner, and loop primers. The document further describes detecting an amplified product by labeled probe hybridization using molecular beacons or intercalating dyes.
The molecular beacons are described as fluorophore/quencher hairpin probes, with probe sequence regions defined as corresponding to specified SEQ ID NOs and length windows. The document also describes example probe sequences using defined SEQ ID NOs and testing cross-reactivity against common human coronaviruses, including the use of a multi-target amplification approach with multiple primer sets in a single reaction well.
Claims Coverage
The partial content includes two independent claims. Claim coverage centers on a specific primer-set composition defined by SEQ ID NOs and a SARS-CoV-2 detection method using nucleic-acid extraction, RTLAMP with a specified Set-13 primer set, and detecting whether an amplified product is present or absent, with dependent coverage for probe-based detection and kit components.
Sequence-specific primer set composition defined by SEQ ID NOs
A composition comprising a set of polynucleotides including a primer set with a forward outer primer comprising SEQ ID NO: 63, a backward outer primer comprising SEQ ID NO: 86, a forward inner primer comprising SEQ ID NO: 65, a backward inner primer comprising SEQ ID NO: 87, a forward loop primer comprising SEQ ID NO: 67, and a backward loop primer comprising SEQ ID NO: 88.
SARS-CoV-2 detection method using RTLAMP with Set-13
A method of detecting SARS-CoV-2 in a test sample comprising extracting nucleic acid, amplifying a target sequence using a reaction mixture comprising a strand displacement DNA polymerase and reverse transcriptase and a sequence-specific primer set wherein the sequence-specific primer set is Set-13 comprising forward outer primer SEQ ID NO: 63, backward outer primer SEQ ID NO: 86, forward inner primer SEQ ID NO: 65, backward inner primer SEQ ID NO: 87, forward loop primer SEQ ID NO: 67, and backward loop primer SEQ ID NO: 88, and detecting the presence or absence of an amplified product, where presence is indicative of SARS-CoV-2.
Overall, the claims in the provided partial content focus on tightly defined primer sequences for RTLAMP, including Set-13, and on detecting amplified products as indicative of SARS-CoV-2, with additional dependent coverage for probe-defined molecular beacon labeling and kit inclusion of enzymatic components.
Stated Advantages
Documented Applications
No documented applications found
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