CHEMILUMINESCENT PROBES FOR MULTIPLEX MOLECULAR QUANTIFICATION AND USES THEREOF
A novel method is disclosed for simultaneous detection and quantification of two or more nucleic acid targets, without need for amplification. The method depends on spectral-temporal resolution of chemiluminescence emitted from independent hybridization-induced chemiluminescent signal (HICS) probes. The utility of this method has been demonstrated by use of resolvable N-linked acridinium and 2,7-dimethoxyacridinium ester labeled probes in a homogeneous assay for sensitive and simultaneous independent quantification of several bacterial and fungal target sequences. Compositions and kits for practicing the method of the present invention are also disclosed.
1 . A homogeneous assay for quantifying at least two target nucleic acid sequences in a sample, said assay comprising the steps of:
(a) sequentially or simultaneously:
(i) contacting the sample with a first hybridization induced chemiluminescent signal (HICS) probe comprising a first chemiluminescent molecule, under conditions allowing hybridization of the first probe to a first target nucleic acid sequence;
(ii) contacting the sample with a second HICS probe comprising a second chemiluminescent molecule, under conditions allowing hybridization of the second probe to a second target nucleic acid sequence that is different from the first target sequence;
(b) chemically triggering the first and second chemiluminescent molecules;
(c) detecting chemiluminescence emissions from the first and second chemiluminescent molecules, wherein the first and second chemiluminescent molecules have sufficiently different emission profiles to allow spectral and/or temporal resolution of the chemiluminescence emissions; and
(d) quantifying the target nucleic acid sequences in the sample based on the chemiluminescence emissions from the first and second chemiluminescent molecules.
2 . The assay of claim 1 , wherein the emission maxima of the first and second chemiluminescent molecules are separated by at least 50 nm.
3 . The assay of claim 1 , wherein the first and second chemiluminescent molecules comprise different acridinium ester (AE) moieties conjugated through an acridinium position other than C9 to a stem-loop oligonucleotide.
4 . The assay of claim 3 , wherein the first chemiluminescent molecule comprises an unsubstituted AE moiety, and the second chemiluminescent molecule comprises a 2,7-substituted AE moiety.
5 . The assay of claim 3 , wherein the AE moieties are conjugated through the N10 acridinium position to a stem-loop oligonucleotide.
6 . The assay of claim 5 , wherein the first chemiluminescent molecule comprises an unsubstituted AE moiety, and the second chemiluminescent molecule comprises a 2,7-substituted AE moiety.
7 . The assay of claim 6 , wherein the unsubstituted AE moiety is a 9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium salt.
8 . The assay of claim 6 , wherein the substituted AE moiety is a 2,7-dimethoxy-9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium salt.
9 . The assay of claim 1 , wherein the target nucleic acid sequences are selected from the group consisting of a pan-bacterial target sequence, a pan-fungal target sequence, a Chlamydia trachomatis ( C. trachomatis ) target sequence and a Neisseria gonorrhoeae ( N. gonorrhoeae ) target sequence.
10 . The assay of claim 9 , wherein the pan-bacterial target sequence comprises an Escherichia coli ( E. coli ) 23S rRNA fragment consisting of SEQ ID NO:1, allowing for a DNA equivalent thereof.
11 . The assay of claim 10 , wherein one of the probes comprises a stem-loop oligonucleotide having a target complementary base sequence consisting of SEQ ID NO:5, allowing for a DNA equivalent thereof.
12 . The assay of claim 11 , wherein the stem-loop oligonucleotide comprises a base sequence consisting of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, allowing for DNA equivalents thereof.
13 . The assay of claim 9 , wherein the pan-fungal target sequence comprises a Candida albicans ( C. albicans ) 18S rRNA fragment consisting of SEQ ID NO:2, allowing for a DNA equivalent thereof.
14 . The assay of claim 13 , wherein the one of the probes comprises a stem-loop oligonucleotide having a target complementary sequence consisting of SEQ ID NO:6, allowing for an RNA equivalent thereof.
15 . The assay of claim 14 , wherein the stem-loop oligonucleotide comprises a base sequence consisting of SEQ ID NO:12 or SEQ ID NO:13, allowing for RNA equivalents thereof.
16 . The assay of claim 9 , wherein the C. trachomatis target sequence comprises a 23S rRNA fragment consisting of SEQ ID NO:3, allowing for a DNA equivalent thereof.
17 . The assay of claim 16 , wherein one of the probes comprises a stem-loop oligonucleotide having a target complementary sequence consisting of SEQ ID NO:7, allowing for a DNA equivalent thereof.
18 . The assay of claim 17 , wherein the stem-loop oligonucleotide comprises a base sequence consisting of SEQ ID NO:14, allowing for a DNA equivalent thereof.
19 . The assay of claim 9 , wherein the N. gonorrhoeae target sequence comprises a 16S rRNA fragment consisting of SEQ ID NO:4, allowing for a DNA equivalent thereof.
20 . The assay of claim 19 , wherein one of the probes comprises a stem-loop oligonucleotide having a target complementary base sequence consisting of SEQ ID NO:8, allowing for a DNA equivalent thereof.
21 . The assay of claim 20 , wherein the stem-loop oligonucleotide comprises a base sequence consisting of SEQ ID NO:15, allowing for a DNA equivalent thereof.
22 . A composition comprising a 2,7-dimethoxy-9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium salt.
23 . The composition of claim 22 , wherein the salt is 2,7-dimethoxy-9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium iodide.
24 . The composition of claim 22 , wherein the 2,7-dimethoxy-9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium salt is directly or indirectly conjugated to a small organic biomolecule, a macromolecule, a viral particle, a sub-cellular component, or a cell.
25 . The composition of claim 24 , wherein the macromolecule is selected from the group consisting of a protein, a peptide, a nucleic acid, an oligonucleotide, a polysaccharide, an oligosaccharide, a glycoprotein, a glycosaminoglycan, a lectin, a lipoprotein, a lipopolysaccharide, a hormone, a toxin, a cytokine, and a combination thereof.
26 . The composition of claim 25 , wherein the oligonucleotide is a stem-loop oligonucleotide.
27 . A kit comprising the composition of claim 22 and reagent means for triggering a chemiluminescence emission.
28 . The kit for detecting and/or quantifying a target nucleic acid sequence in a sample, the kit comprising the composition of claim 26 and reagent means for triggering a chemiluminescence emission.
29 . A kit for detecting and/or quantifying at least two target nucleic acid sequences in a sample, the kit comprising the probes according to claim 6 and reagent means triggering a chemiluminescence emission.
30 . The kit of claim 29 , wherein the unsubstituted AE moiety is a 9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium salt.
31 . The kit of claim 29 , wherein the substituted AE moiety is a 2,7-dimethoxy-9-(2,6-dibromophenoxycarbonyl)-10-(3-carbonylpropyl)acridinium salt.