Ultra sensitive method for in situ detection of nucleic acids

Abstract

Disclosed is a method for in situ detection of one or more target nucleic acids based on a combination of RNAscope® 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 RNAscope® assay and a general ISH signal amplification assay, for sensitive detection of one or more target nucleic acids.

What is claimed is: 
     
         1 . 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 a signal generating multimer capable of hybridizing to said set of two or more capture probes, wherein said signal generating multimer comprises a label probe;   (d) providing a signal amplification probe capable of binding to said label probe, wherein said signal amplification probe comprises a label;   (e) hybridizing said target nucleic acid to said set of two or more capture probes;   (f) capturing the signal generating multimer to said set of two or more capture probes and thereby capturing the signal generating multimer to said target nucleic acid;   (g) capturing said signal amplification probe to said label probe and thereby capturing the signal amplification probe to said signal generating multimer; and   (h) detecting the presence, absence, or amount of the label.   
     
     
         2 . The method of  claim 1 , wherein said signal amplification probe comprises: a biotin molecule which is capable of conjugating to said label probe, an avidin/streptavidin molecule which is capable of binding to said biotin molecule, and additional biotin molecules being conjugated to Horse Radish Peroxidase (HRP), Alkaline Phosphatase (AP), or fluorophores and are capable of binding to said avidin/streptavidin molecule. 
     
     
         3 . The method of  claim 1 , wherein said signal amplification probe comprises: a HRP, AP, Dinitrophenyl (DNP), or fluorophore molecule which is capable of conjugating to said label probe, one or more first antibody which is capable of binding to said HRP, AP, DNP, or fluorophore molecule, and one or more second antibody being conjugated to HRP, Polymer-HRP, AP, Polymer-AP or fluorophore and is capable of binding to said one or more first antibody. 
     
     
         4 . The method of  claim 1 , wherein said signal amplification probe comprises: a HRP molecule which is capable of conjugating to said label probe, a plurality of tyramide-biotin or tyramide-fluorophore molecules which are capable of reacting with said HRP molecule, and detection labels which are capable detecting said tyramide-biotin or tyramide-fluorophore molecules visually. 
     
     
         5 . The method of  claim 4 , wherein said detection labels are a combination of avidin/streptavidin-HRP and a chromogenic substrate or a combination of avidin/streptavidin-AP and a chromogenic substrate. 
     
     
         6 . The method of  claim 5 , wherein said chromogenic substrate is selected from the group consisting of: diaminobenzine (DAB) and Fast Red. 
     
     
         7 . The method of  claim 4 , wherein said signal generating multimer comprises either (i) said label probe capable of hybridizing to said set of two or more capture probes, (ii) said label probe and an amplifier hybridized to the label probe and capable of hybridizing to said set of two or more capture probes, and (iii) said label probe, an amplifier hybridized to the label, and a preamplifier hybridized to the amplifier and capable of hybridizing to said set of two or more capture probes. 
     
     
         8 . The method of  claim 7 , wherein the preamplifier:amplifier ratio is between 1-16, between 2-10, or between 2-5. 
     
     
         9 . The method of  claim 8 , wherein said target nucleic acid is selected from the group consisting of: a DNA, a cDNA, an RNA, and an mRNA. 
     
     
         10 . The method of  claim 8 , wherein step (a) comprises capturing said target nucleic acid on a solid support. 
     
     
         11 . The method of  claim 8 , wherein in step (a), said sample comprises a cell comprising or suspected of comprising the target nucleic acid. 
     
     
         12 . The method of  claim 8 , wherein in step (a), said sample comprises a cell comprising or suspected of comprising two or more different target nucleic acids. 
     
     
         13 . The method of  claim 8 , wherein in step (a), said sample comprises two or more different cells, each comprising or suspected of comprising a different target nucleic acid. 
     
     
         14 . The method of  claim 12  or  13 , wherein the two different target nucleic acids are detected using a dual-color chromogenic in situ hybridization (CISH) or a dual-color fluorescent in situ hybridization (FISH). 
     
     
         15 . The method of  claim 14 , wherein said dual-color CISH is performed using two different signal amplification probes, wherein the first signal amplification probe comprises: tyramide-biotin, streptavidin-HRP and DAB, and the second signal amplification probe comprises: anti-DNP-AP and Fast Red. 
     
     
         16 . A kit comprising:
 (a) a target nucleic acid;   (b) one set of two or more capture probes capable of hybridizing to said target nucleic acid;   (c) a signal generating multimer capable of hybridizing to said set of two or more capture probes, wherein said signal generating multimer comprises a label probe; and   (d) a signal amplification probe capable of binding to said label probe, wherein said signal amplification probe comprises a label.   
     
     
         17 . The kit of  claim 16 , wherein said signal amplification probe comprises: a biotin molecule which is capable of conjugating to said label probe, an avidin/streptavidin molecule which is capable of binding to said biotin molecule, and additional biotin molecules being conjugated to HRP, AP or fluorophores and are capable of binding to said avidin/streptavidin molecule. 
     
     
         18 . The kit of  claim 16 , wherein said signal amplification probe comprises: a HRP, AP, DNP, or fluorophore molecule which is capable of conjugating to said label probe, one or more first antibody which is capable of binding to said HRP, AP, DNP, or fluorophore molecule, and one or more second antibody being conjugated to HRP, Polymer-HRP, AP, Polymer-AP or fluorophore and is capable of binding to said one or more first antibody. 
     
     
         19 . The kit of  claim 16 , wherein said signal amplification probe comprises: a HRP molecule which is capable of conjugating to said label probe, a plurality of tyramide-biotin or tyramide-fluorophoremolecules which are capable of reacting with said HRP molecule, and detection labels which are capable detecting said tyramide-biotin molecules visually. 
     
     
         20 . The kit of  claim 19 , wherein said detection labels are a combination of avidin/streptavidin-HRP and a chromogenic substrate or a combination of avidin/streptavidin-AP and a chromogenic substrate. 
     
     
         21 . The method of  claim 20 , wherein said chromogenic substrate is selected from the group consisting of: DAB and Fast Red. 
     
     
         22 . The kit of  claim 19 , wherein said signal generating multimer comprises either (i) said label probe capable of hybridizing to said set of two or more capture probes, (ii) said label probe and an amplifier hybridized to the label probe and capable of hybridizing to said set of two or more capture probes, and (iii) said label probe, an amplifier hybridized to the label, and a preamplifier hybridized to the amplifier and capable of hybridizing to said set of two or more capture probes. 
     
     
         23 . The method of  claim 22 , wherein the preamplifier:amplifier ratio is between 1-16, between 2-10, or between 2-5. 
     
     
         24 . The kit of  claim 23 , wherein said target nucleic acid is selected from the group consisting of: a DNA, a cDNA, an RNA, and an mRNA. 
     
     
         25 . The kit of  claim 23 , further comprising a cell comprising or suspected of comprising the target nucleic acid. 
     
     
         26 . The kit of  claim 23 , further comprising two or more different cells, each comprising or suspected of comprising a different target nucleic acid. 
     
     
         27 . The kit of  claim 25  or  26 , wherein said signal amplification probe is either a dual-color chromogenic in situ hybridization (CISH) probe or a dual-color fluorescent in situ hybridization (FISH) probe. 
     
     
         28 . The kit of  claim 27 , wherein in said dual-color CISH probe, the first signal amplification probe comprises: tyramide-biotin, streptavidin-HRP and DAB, and the second signal amplification probe comprises: anti-DNP-AP and Fast Red.