Dengue virus assay

Abstract

Nucleic acid assays for detecting nucleic acids of Dengue virus serotypes 1-4.

1 - 23 . (canceled) 
     
     
         24 . A composition for amplifying the nucleic acids of Dengue virus serotypes 1-4 in a nucleic acid amplification reaction, said composition comprising:
 at least one first-strand priming oligonucleotide, the base sequence of said at least one first-strand priming oligonucleotide consisting of a target-hybridizing sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:10, optionally joined to an upstream promoter sequence, and further optionally joined to an upstream tag sequence, wherein neither the optional upstream promoter sequence nor the optional tag sequence can hybridize to any Dengue virus nucleic acid and participate in said nucleic acid amplification reaction in the absence of joining to said target-hybridizing sequence; and   at least one second-strand priming oligonucleotide, the base sequence of one of said at least one second-strand priming oligonucleotides consisting of SEQ ID NO:13, wherein said composition detectably amplifies nucleic acids of Dengue virus serotypes 1-4 when present in amounts as low as 10 copies per 100 μl of reaction volume at the start of said nucleic acid amplification reaction.   
     
     
         25 . The composition of  claim 24 , wherein said at least one first-strand priming oligonucleotide comprises two first-strand priming oligonucleotides, the base sequence of one of the two first-strand priming oligonucleotides consisting of SEQ ID NO:1, optionally joined to said upstream promoter sequence, and the base sequence of the other of the two first-strand priming oligonucleotides consisting of SEQ ID NO:10, optionally joined to said upstream promoter sequence. 
     
     
         26 . The composition of  claim 25 , wherein each of the two first-strand priming oligonucleotides includes said upstream promoter sequence. 
     
     
         27 . The composition of  claim 26 , wherein said at least one second-strand priming oligonucleotide further comprises an additional second-strand priming oligonucleotide, the base sequence of said additional second-strand priming oligonucleotide consisting of SEQ ID NO:16. 
     
     
         28 . The composition of  claim 24 , wherein said at least one second-strand priming oligonucleotide further comprises an additional second-strand priming oligonucleotide, the base sequence of said additional second-strand priming oligonucleotide consisting of SEQ ID NO:16. 
     
     
         29 . A kit for use in detecting the nucleic acids of each of Dengue virus serotypes 1-4 in a nucleic acid amplification reaction, said kit comprising in packaged combination of one of more containers:
 at least one first-strand priming oligonucleotide, the base sequence of said at least one first-strand priming oligonucleotide consisting of a target-hybridizing base sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:10, optionally joined to an upstream promoter sequence, and further optionally joined to an upstream tag sequence, wherein neither the upstream promoter sequence nor the tag sequence hybridizes to any Dengue virus nucleic acid and participates in said nucleic acid amplification reaction in the absence of joining to said target-hybridizing base sequence; and   a second oligonucleotide, the base sequence of said second oligonucleotide consisting of SEQ ID NO:19, allowing for RNA and DNA equivalent base substitutions.   
     
     
         30 . The kit of  claim 29 , wherein the base sequence of said at least one first-strand priming oligonucleotide consists of SEQ ID NO:1 joined to both said upstream promoter sequence and said upstream tag sequence, wherein said upstream tag sequence is positioned between the target-hybridizing base sequence of said at least one first-strand priming oligonucleotide, and wherein said second oligonucleotide is a priming oligonucleotide that comprises a DNA backbone. 
     
     
         31 . The kit of  claim 30 , further comprising a tagged promoter oligonucleotide consisting of said tag sequence joined to said upstream promoter sequence. 
     
     
         32 . The kit of  claim 29 , further comprising a second-strand priming oligonucleotide, the base sequence of said second-strand priming oligonucleotide consisting of SEQ ID NO:13, wherein said second oligonucleotide is a hybridization detection probe, and wherein said at least one first-strand priming oligonucleotide and said second-strand priming oligonucleotide, when combined in said nucleic acid amplification reaction detectably amplify nucleic acids of Dengue virus serotypes 1-4 when present in amounts as low as 10 copies per 100 μl of reaction volume at the start of said nucleic acid amplification reaction. 
     
     
         33 . The kit of  claim 32 , further comprising an additional second-strand priming oligonucleotide, the base sequence of said additional second-strand priming oligonucleotide consisting of SEQ ID NO:16. 
     
     
         34 . The kit of  claim 29 , wherein said at least one first-strand priming oligonucleotide comprises two first-strand priming oligonucleotides, the base sequence of one of the two first-strand priming oligonucleotides consisting of SEQ ID NO:1, optionally joined to said upstream promoter sequence, and the base sequence of the other of the two first-strand priming oligonucleotides consisting of SEQ ID NO:10, optionally joined to said upstream promoter sequence. 
     
     
         35 . The kit of  claim 34 , wherein each of the two first-strand priming oligonucleotides includes said upstream promoter sequence. 
     
     
         36 . The kit of  claim 35 , further comprising a second-strand priming oligonucleotide, the base sequence of said second-strand priming oligonucleotide consisting of SEQ ID NO:13, and an additional second-strand priming oligonucleotide, the base sequence of said additional second-strand priming oligonucleotide consisting of SEQ ID NO:16, wherein said second oligonucleotide with the base sequence of SEQ ID NO:19 is a hybridization detection probe. 
     
     
         37 . The kit of  claim 30 , further comprising a probe oligonucleotide. 
     
     
         38 . The kit of  claim 37 , wherein the base sequence of said probe oligonucleotide consists of SEQ ID NO:25. 
     
     
         39 . The kit of  claim 34 , further comprising a second-strand priming oligonucleotide, the base sequence of said second-strand priming oligonucleotide consisting of SEQ ID NO:13, and wherein said second oligonucleotide is a hybridization detection probe. 
     
     
         40 . A kit for use in detecting the nucleic acids of each of Dengue virus serotypes 1-4 in a nucleic acid amplification reaction, said kit comprising in packaged combination of one of more containers:
 at least one first-strand priming oligonucleotide up to 73 bases in length, the 3′ terminal base sequence of said at least one first-strand priming oligonucleotide consisting of a target-hybridizing base sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:10, and SEQ ID NOs:42-47 optionally joined to an upstream promoter sequence, and further optionally joined to an upstream tag sequence, wherein neither the upstream promoter sequence nor the upstream tag sequence hybridizes to any Dengue virus nucleic acid and participates in said nucleic acid amplification reaction in the absence of joining to said target-hybridizing base sequence; and   at least one second-strand priming oligonucleotide that hybridizes to an extension product of said at least one first-strand priming oligonucleotide using any of SEQ ID NOs:36-39 as a template, wherein the first-strand and second-strand priming oligonucleotides in combination detectably amplify nucleic acids of Dengue virus serotypes 1-4 when present in amounts as low as 10 copies per 100 μl of reaction volume at the start of said nucleic acid amplification reaction.   
     
     
         41 . The kit of  claim 40 , wherein the base sequence of said at least one second-strand priming oligonucleotide consists of SEQ ID NO:19, allowing for RNA and DNA equivalent base substitutions. 
     
     
         42 . The kit of  claim 40 , wherein the base sequence of said at least one second-strand priming oligonucleotide consists of SEQ ID NO:13. 
     
     
         43 . The kit of  claim 40 , wherein the base sequence of said at least one second-strand priming oligonucleotide consists of SEQ ID NO:16. 
     
     
         44 . The kit of  claim 40 , wherein position 10 of said target-hybridizing base sequence of said at least one first-strand priming oligonucleotide is inosine. 
     
     
         45 . A kit for use in detecting the nucleic acids of Dengue virus serotype 3 in a nucleic acid amplification reaction, said kit comprising in packaged combination of one of more containers:
 at least one first-strand priming oligonucleotide up to 73 bases in length, the 3′ terminal base sequence of said at least one first-strand priming oligonucleotide consisting of a target-hybridizing base sequence selected from the group consisting of SEQ ID NOs:48-55 optionally joined to an upstream promoter sequence, and further optionally joined to an upstream tag sequence, wherein neither the upstream promoter sequence nor the tag sequence hybridizes to any Dengue virus nucleic acid and participates in said nucleic acid amplification reaction in the absence of joining to said target-hybridizing base sequence; and   at least one second-strand priming oligonucleotide that hybridizes to an extension product of said at least one first-strand priming oligonucleotide using any of SEQ ID NOs:38 as a template, wherein the first-strand and second-strand priming oligonucleotides in combination detectably amplify nucleic acids of Dengue virus serotype 3 when present in amounts as low as 10 copies per 100 μl of reaction volume at the start of said nucleic acid amplification reaction.   
     
     
         46 . The kit of  claim 45 , wherein the base sequence of said at least one second-strand priming oligonucleotide consists of SEQ ID NO:19, allowing for RNA and DNA equivalent base substitutions. 
     
     
         47 . The kit of  claim 45 , wherein the base sequence of said at least one second-strand priming oligonucleotide consists of SEQ ID NO:13. 
     
     
         48 . The kit of  claim 45 , wherein the base sequence of said at least one second-strand priming oligonucleotide consists of SEQ ID NO:16. 
     
     
         49 . The kit of  claim 45 , wherein position 10 of said target-hybridizing base sequence of said at least one first-strand priming oligonucleotide is inosine.