RNA DETECTION METHOD
First Claim
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1. A method for the detection of a target RNA sequence in a sample comprising the steps of:
- (a) contacting single-stranded RNA (ssRNA) molecules present in a sample with a labelled oligonucleotide containing a sequence substantially complementary to a region of the target RNA under hybridisation conditions to provide a mixture of RNA-oligonucleotide duplexes wherein the RNA-oligonucleotide duplexes comprise the target RNA annealed to the labelled oligonucleotide;
(b) maintaining the mixture of step (a) with an RNA-dependent RNA polymerase (RdRp) under RNA polymerisation conditions in the absence of a primer wherein said RdRp has an RNA-oligonucleotide duplex separation activity and is capable of de novo RNA synthesis such that the RdRp polymerises an RNA strand complementary to the ssRNA in the sample and releases the labelled oligonucleotide; and
(c) detecting and/or measuring the signal generated by the released labelled oligonucleotide.
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Abstract
The present invention relates to methods for the detection of target RNA sequences and to RNA amplification methods making use of strand displacement techniques employing RNA-dependent RNA polymerases having RNA-oligonucleotide duplex separation activity and being capable of de novo RNA synthesis in the absence of a primer. The present invention further relates to kits for carrying out such methods.
5 Citations
21 Claims
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1. A method for the detection of a target RNA sequence in a sample comprising the steps of:
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(a) contacting single-stranded RNA (ssRNA) molecules present in a sample with a labelled oligonucleotide containing a sequence substantially complementary to a region of the target RNA under hybridisation conditions to provide a mixture of RNA-oligonucleotide duplexes wherein the RNA-oligonucleotide duplexes comprise the target RNA annealed to the labelled oligonucleotide; (b) maintaining the mixture of step (a) with an RNA-dependent RNA polymerase (RdRp) under RNA polymerisation conditions in the absence of a primer wherein said RdRp has an RNA-oligonucleotide duplex separation activity and is capable of de novo RNA synthesis such that the RdRp polymerises an RNA strand complementary to the ssRNA in the sample and releases the labelled oligonucleotide; and (c) detecting and/or measuring the signal generated by the released labelled oligonucleotide. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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2. An RNA amplification method comprising the steps of:
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(i) contacting a single-stranded RNA (ssRNA) template with a labelled oligonucleotide containing a sequence substantially complementary to a region of said ssRNA under hybridisation conditions to provide RNAoligonucleotide duplexes comprising the ssRNA annealed to the labelled oligonucleotide; (ii) maintaining the RNA-oligonucleotide duplexes of step (i) with an RNA-dependent RNA polymerase (RdRp) under RNA polymerisation conditions in the absence of a primer wherein said RdRp has an RNA-oligonucleotide duplex separation activity and is capable of de novo RNA synthesis such that the RdRp polymerises an RNA strand complementary to the ssRNA and releases the labelled oligonucleotide; (iii) separating the RNA duplexes formed by the RdRp between the ssRNA and the polymerised complementary strand; (iv) optionally, repeating steps (i) to (iii); and (v) detecting and/or measuring the signal generated by the released labelled oligonucleotide.
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14. A kit for detecting a target RNA sequence in a sample comprising:
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at least one labelled oligonucleotide containing a sequence substantially complementary to a region of the target RNA sequence; an RNA-dependent RNA polymerase (RdRp) having an RNA duplex separation activity under polymerisation conditions and being capable of de novo RNA synthesis in the absence of a primer. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21)
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Specification