Ultra sensitive method for in situ detection of nucleic acids
AssigneeAdvanced Cell Diagnostics, Inc.
InventorsXingyong Wu, Huei-Yu Fay Wang, Nan Su, Li-Chong Wang, Yuling Luo
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Abstract
Disclosed is a method for in situ detection of one or more target nucleic acids based on a combination of an in situ hybridization (ISH) assay method and a general ISH signal amplification method. This new method produces high signal intensity and while keeps low background noise of signal amplification. The result can be consistently reproduced and the method can be easily adopted for routine clinic diagnostic use. Further, the invention relates to a kit, comprising the components of the ISH assay and a general ISH signal amplification assay, for sensitive detection of one or more target nucleic acids.
Claims29
A method of detecting at least one target nucleic acid, the method comprising:
- (a) providing a sample comprising or suspected of comprising said target nucleic acid;
- (b) providing at least one set of two or more capture probes capable of hybridizing to said target nucleic acid;
- (c) providing:
- (i) an amplifier capable of hybridizing to a label probe;
- (ii) a preamplifier capable of hybridizing to the amplifier and capable of hybridizing to said set of two or more capture probes, wherein the preamplifier:amplifier ratio is between 1:1-16, between 1:2-10, or between 1:2-5;
- (iii) one or more label probes, wherein each label probe is conjugated to a biotin molecule or a hapten molecule;
- and
- (iv) (1) an avidin/streptavidin molecule capable of binding to said biotin molecule, and additional biotin molecules conjugated to horse radish peroxidase (HRP), alkaline phosphatase (AP), or fluorophore, wherein said additional biotin molecules are capable of binding to said avidin/streptavidin molecule;
- or
- (2) one or more first antibodies capable of binding to said hapten molecule, and one or more second antibodies conjugated to horse radish peroxidase (HRP), alkaline phosphatase (AP), or fluorophore, wherein said one or more second antibodies are capable of binding to said one or more first antibodies;
- (d) hybridizing said set of two or more capture probes to said target nucleic acid;
- (e) capturing the preamplifier, amplifier and label probe to said set of two or more capture probes, thereby capturing the label probe to said target nucleic acid, wherein the label probe is bound to said avidin/streptavidin bound to said additional biotin molecules conjugated to HRP, AP or fluorophore, or wherein the label probe is bound to said hapten bound to said one or more first antibodies bound to said one or more second antibodies conjugated to HRP, AP or fluorophore;
- and
- (f) detecting the presence, absence, or amount of the HRP, AP or fluorophore associated with the captured label probe.
A kit comprising:
- (a) at least one set of two or more capture probes capable of hybridizing to a target nucleic acid;
- (b) an amplifier capable of hybridizing to a label probe;
- (c) a preamplifier capable of hybridizing to the amplifier and capable of hybridizing to said set of two or more capture probes, wherein the preamplifier:amplifier ratio is used between 1:1-16, between 1:2-10, or between 1:2-5;
- (d) one or more label probes, wherein each label probe is conjugated to a biotin molecule or a hapten molecule;
- and
- (e) (1) an avidin/streptavidin molecule capable of binding to said biotin molecule, and additional biotin molecules conjugated to horse radish peroxidase (HRP), alkaline phosphatase (AP), or fluorophore, wherein said additional biotin molecules are capable of binding to said avidin/streptavidin molecule;
- or
- (2) one or more first antibodies capable of binding to said hapten molecule, and one or more second antibodies conjugated to horse radish peroxidase (HRP), alkaline phosphatase (AP) or fluorophore, wherein said one or more second antibodies are capable of binding to said one or more first antibodies.
Dependencies are inferred from the claim text.