Polynucleotides for amplification and detection of human beta actin
Inventors
Casolari, Jason M. • Jiang, Xuewen • Maamar, Hédia
Assignees
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Publication Number
US-11891662-B2
Publication Date
2024-02-06
Expiration Date
Abstract
Disclosed herein are primers and probes related to the detection of beta actin [Homo sapiens (human)] via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of β-actin present in test samples. Specifically, the present disclosure describes primers and probes that bind to the beta actin gene for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.
Core Innovation
The disclosure relates to NAAT detection of human β-actin using loop mediated isothermal amplification (LAMP) with sequence-specific primer sets. It specifies primer sets for β-actin, including set-29, and provides set-29 primer sequences associated with SEQ ID NO: 132 and SEQ ID NO: 133. Detection is performed by identifying amplified products resulting from the isothermal LAMP reaction.
The disclosure further provides fluorescence detection using molecular beacon probes. The molecular beacon probes include a fluorophore and a quencher and are described with a molecular beacon hairpin architecture, including SEQ ID NOs 135-184 with SEQ ID NO: 184 identified in particular. Preferred labeling is FAM/BHQ1, and alternatives such as BHQ2, ATTO 565, and Alexa 594 are also described for fluorophore and quencher combinations.
The disclosure describes rapid isothermal amplification using a strand displacement DNA polymerase, including conditions centered on an isothermal temperature range of about 60-67°C and rapid run times. It also describes RT-LAMP using optional reverse transcriptase (RTx) for nucleic acid processing when applicable. The document includes kit compositions containing primer sets and molecular beacon probes, optionally including polymerase and/or RT, and provides example results including time-to-positive comparisons across primer sets and molecular-beacon-based detection.
Claims Coverage
The independent claims cover three inventive features: set-29 primer composition, molecular beacon probe identity, and rapid β-actin detection by LAMP.
Set-29 primer composition with SEQ ID NO: 132 and SEQ ID NO: 133
A composition comprising a set of primers comprising set-29, wherein set-29 comprises primer sequences of SEQ ID NO: 132 and SEQ ID NO: 133.
Molecular beacon probe sequence SEQ ID NO: 184 with fluorophore and quencher
A molecular beacon probe comprising a fluorophore and a quencher, wherein the probe sequence comprises SEQ ID NO: 184.
Rapid β-actin detection using strand displacement DNA polymerase and sequence-specific LAMP primer set set-29
A method of detecting β-actin in a test sample by extracting nucleic acid, amplifying a target sequence by reacting nucleic acid for less than ten minutes with a reaction mixture comprising a strand displacement DNA polymerase and a sequence-specific isothermal loop mediated amplification (LAMP) primer set, and detecting presence or absence of an amplified product indicative of β-actin, wherein the sequence-specific LAMP primer set is set-29 comprising primer sequences of SEQ ID NO: 132 and SEQ ID NO: 133.
Overall, the claim coverage centers on set-29 primer identity, a specific molecular beacon probe identity with fluorophore and quencher, and a β-actin detection method combining nucleic acid extraction, rapid isothermal LAMP using strand displacement DNA polymerase with set-29, and amplified-product presence or absence detection.
Stated Advantages
Documented Applications
No documented applications found
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