Sequence Specific Real-Time Monitoring of Loop-Mediated Isothermal Amplification (LAMP)
AssigneeUniversity of Hawaii
InventorsRyo Kubota, Daniel M. Jenkins
Life of this patent
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Abstract
Gene-based diagnostics capable of rapidly discriminating selected strains of a selected pathogen from other populations within the same species are disclosed. Sequence-specific, real-time monitoring of LAMP of DNA may be accomplished through the use of oglionucleotide probes, referred to as “assimilating probes.” The assimilating probes include two oglionucleotide strands, one which includes a quencher (referred to as the quenching probe) and another which includes a fluorophore (referred to as the fluorescent probe). A fluorescent signal results when the two strands are displaced from one another during the LAMP reaction. By monitoring the emitted fluorescence, sequence specific amplification may be detected.
Claims21
A method of monitoring LOOP-mediated isothermal amplification (LAMP) of a target DNA, comprising:
- contacting an assimilating probe and a DNA polymerase with a LAMP reaction mixture,
- wherein the LAMP reaction mixture comprises the target DNA and one or more LAMP primers that hybridize with the target DNA,
- wherein the assimilating probe comprises a first and second oligonucleotide strands, wherein the first oligonucleotide strand comprises a quencher probe at a 3′ end and wherein the second oligonucleotide strand of the assimilating probe comprises a fluorophore at a 5′ end;
- wherein the ratio of the amount of the second oligonucleotide strand to the amount of the first oligonucleotide strand is less than 1:1;
- and
- measuring fluorescence emitted by the LAMP reaction mixture that has been contacted with the assimilating probe and the DNA polymerase.
(canceled)
An assimilating probe for monitoring LOOP-mediated isothermal amplification (LAMP) of a target DNA comprising:
- a first oligonucleotide strand comprising a quencher probe at a 3′ end of the first oligonucleotide strand;
- and
- a second oligonucleotide strand comprising a fluorophore at a 5′ end of the second oligonucleotide strand;
- wherein the ratio of the amount of the second oligonucleotide strand to the first oligonucleotide strand is less than 1:1.
A method of detecting the presence or absence of a target DNA
- contacting an assimilating probe and a DNA polymerase with a LAMP reaction mixture,
- wherein the LAMP reaction mixture comprises a sample to be tested for the presence of a target DNA and one or more LAMP primers capable of amplifying the target DNA,
- wherein the assimilating probe comprises a first and second oligonucleotide strands, wherein the first oligonucleotide strand comprises a quencher probe at a 3′ end and wherein the second oligonucleotide strand of the assimilating probe comprises a fluorophore at a 5′ end;
- wherein the ratio of the amount of the second oligonucleotide strand to the amount of the first oligonucleotide strand is less than 1:1;
- and
- detecting the presence or absence of the target DNA.
Dependencies are inferred from the claim text.