Methods and reagents for detection of chikungunya virus and zika virus

Abstract

Methods and oligonucleotide reagents for diagnosing chikungunya virus and Zika virus infections are described. In particular, the invention relates to quantitative assays that can detect all lineages of chikungunya virus and Zika virus and distinguish chikungunya virus and Zika virus from each other as well as dengue virus and other arbovirus pathogens.

1 . A composition for detecting chikungunya virus in a biological sample using a nucleic acid amplification assay, the composition comprising at least one set of oligonucleotide primers comprising a forward primer and a reverse primer capable of amplifying at least a portion of a chikungunya virus genome, wherein said primers are not more than 40 nucleotides in length, wherein said set of primers is selected from the group consisting of:
 a) a forward primer comprising the nucleotide sequence of SEQ ID NO:6 and a reverse primer comprising the sequence of SEQ ID NO:7;   b) a forward primer comprising at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:6 and a reverse primer comprising at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:7;   c) a forward primer comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:6 and a reverse primer comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:7, wherein the primer is capable of hybridizing to and amplifying chikungunya virus nucleic acids in the nucleic acid amplification assay;   d) a forward primer and a reverse primer comprising at least one nucleotide sequence that differs from the corresponding nucleotide sequence of the forward primer or reverse primer of the primer set of (a) in that the primer has up to three nucleotide changes compared to the corresponding sequence, wherein the primer is capable of hybridizing to and amplifying chikungunya virus nucleic acids in the nucleic acid amplification assay; and   e) a forward primer and a reverse primer comprising nucleotide sequences that are complements of the corresponding nucleotide sequences of the forward primer and reverse primer of a primer set selected from the group consisting of (a)-(d).   
     
     
         2 . The composition of  claim 1 , further comprising at least one detectably labeled oligonucleotide probe sufficiently complementary to and capable of hybridizing with a chikungunya virus RNA or an amplicon thereof. 
     
     
         3 . The composition of  claim 2 , wherein the probe is selected from the group consisting of:
 a) a probe comprising the sequence of SEQ ID NO:8;   b) a probe comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:8, wherein the probe is capable of hybridizing to and detecting the chikungunya virus RNA or an amplicon thereof; and   c) a probe that differs from the corresponding nucleotide sequence of SEQ ID NO:8 by up to three nucleotide changes, wherein the probe is capable of hybridizing to and detecting the chikungunya virus RNA or an amplicon thereof.   
     
     
         4 . The composition of  claim 2 , wherein the detectably labeled probe comprises a fluorophore. 
     
     
         5 . The composition of  claim 4 , wherein the detectably labeled probe comprises a 5′- fluorophore and a 3′-quencher. 
     
     
         6 - 74 . (canceled) 
     
     
         75 . A composition for detecting Zika virus in a biological sample using a nucleic acid amplification assay, the composition comprising at least one set of oligonucleotide primers comprising a forward primer and a reverse primer capable of amplifying at least a portion of a Zika virus genome, wherein said primers are not more than 40 nucleotides in length, wherein said set of primers is selected from the group consisting of:
 a) a forward primer comprising the nucleotide sequence of SEQ ID NO:27 and a reverse primer comprising the sequence of SEQ ID NO:28;   b) a forward primer comprising the nucleotide sequence of SEQ ID NO:27 and a reverse primer comprising the sequence of SEQ ID NO:29;   c) a forward primer comprising the nucleotide sequence of SEQ ID NO:27, a first reverse primer comprising the sequence of SEQ ID NO:28, and a second reverse primer comprising the sequence of SEQ ID NO:29;   d) a forward primer comprising at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:27 and at least one reverse primer comprising at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29;   e) a forward primer comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:27 and at least one reverse primer comprising a nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29, wherein the primer is capable of hybridizing to and amplifying Zika virus nucleic acids in the nucleic acid amplification assay;   f) a forward primer and a reverse primer comprising at least one nucleotide sequence that differs from the corresponding nucleotide sequence of the forward primer or reverse primer of the primer set of (a) or (b) in that the primer has up to three nucleotide changes compared to the corresponding sequence, wherein the primer is capable of hybridizing to and amplifying Zika virus nucleic acids in the nucleic acid amplification assay; and   g) a forward primer and a reverse primer comprising nucleotide sequences that are complements of the corresponding nucleotide sequences of the forward primer and reverse primer of a primer set selected from the group consisting of (a)-(f).   
     
     
         76 . The composition of  claim 75 , further comprising at least one detectably labeled oligonucleotide probe sufficiently complementary to and capable of hybridizing with a Zika virus RNA or an amplicon thereof. 
     
     
         77 . The composition of  claim 76 , wherein the probe is selected from the group consisting of:
 a) a probe comprising the sequence of SEQ ID NO:30;   b) a probe comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:30, wherein the probe is capable of hybridizing to and detecting the Zika virus RNA or an amplicon thereof; and   c) a probe that differs from the corresponding nucleotide sequence of SEQ ID NO:30 by up to three nucleotide changes, wherein the probe is capable of hybridizing to and detecting the Zika virus RNA or an amplicon thereof.   
     
     
         78 . The composition of  claim 76 , wherein the detectably labeled probe comprises a fluorophore. 
     
     
         79 . The composition of  claim 78 , wherein the detectably labeled probe comprises a 5′- fluorophore and a 3′-quencher. 
     
     
         80 - 85 . (canceled) 
     
     
         86 . A method for detecting Zika virus, the method comprising:
 a) contacting nucleic acids of a biological sample suspected of containing Zika virus with the composition of  claim 75 ;   b) amplifying at least a portion of a Zika virus RNA, if present; and   c) detecting the presence of the amplified nucleic acids using at least one detectably labeled oligonucleotide probe sufficiently complementary to and capable of hybridizing with the Zika virus RNA or amplicon thereof, if present, as an indication of the presence or absence of Zika virus in the sample.   
     
     
         87 . The method of  claim 86 , wherein the probe is selected from the group consisting of:
 a) a probe comprising the sequence of SEQ ID NO:30;   b) a probe comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:30, wherein the probe is capable of hybridizing to and detecting the Zika virus RNA or an amplicon thereof; and   c) a probe that differs from the corresponding nucleotide sequence of SEQ ID NO:30 by up to three nucleotide changes, wherein the probe is capable of hybridizing to and detecting the Zika virus RNA or an amplicon thereof.   
     
     
         88 . The method of  claim 86 , wherein the detectably labeled probe comprises a fluorophore. 
     
     
         89 . The method of  claim 88 , wherein the detectably labeled probe comprises a 5′- fluorophore and a 3′-quencher. 
     
     
         90 . The method of  claim 89 , wherein the 5′-fluorophore is a fluorescein or rhodamine derivative. 
     
     
         91 - 124 . (canceled) 
     
     
         125 . A kit for detecting Zika virus in a biological sample, the kit comprising: written instructions for identifying the presence of Zika virus; and at least one set of primers comprising a forward primer and a reverse primer capable of amplifying at least a portion of a Zika virus genome, wherein said primers are not more than about 40 nucleotides in length, wherein said set of primers is selected from the group consisting of:
 a) a forward primer comprising the nucleotide sequence of SEQ ID NO:27 and a reverse primer comprising the sequence of SEQ ID NO:28;   b) a forward primer comprising the nucleotide sequence of SEQ ID NO:27 and a reverse primer comprising the sequence of SEQ ID NO:29;   c) a forward primer comprising the nucleotide sequence of SEQ ID NO:27, a reverse primer comprising the sequence of SEQ ID NO:28, and a reverse primer comprising the sequence of SEQ ID NO:29;   d) a forward primer comprising at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:27 and at least one reverse primer comprising at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29;   e) a forward primer comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:27 and at least one reverse primer comprising a nucleotide sequence having at least 95% identity to a sequence selected from the group consisting of SEQ ID NO:28 and SEQ ID NO:29, wherein the primer is capable of hybridizing to and amplifying Zika virus nucleic acids in the nucleic acid amplification assay;   f) a forward primer and a reverse primer comprising at least one nucleotide sequence that differs from the corresponding nucleotide sequence of the forward primer or reverse primer of the primer set of (a) or (b) in that the primer has up to three nucleotide changes compared to the corresponding sequence, wherein the primer is capable of hybridizing to and amplifying Zika virus nucleic acids in the nucleic acid amplification assay; and   g) a forward primer and a reverse primer comprising nucleotide sequences that are complements of the corresponding nucleotide sequences of the forward primer and reverse primer of a primer set selected from the group consisting of (a)-(f).   
     
     
         126 . The kit of  claim 125 , further comprising at least one probe for detecting Zika virus in a biological sample, wherein the probe is selected from the group consisting of:
 a) a probe comprising the sequence of SEQ ID NO:30;   b) a probe comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:30, wherein the probe is capable of hybridizing to and detecting the Zika virus RNA or an amplicon thereof; and   c) a probe that differs from the corresponding nucleotide sequence of SEQ ID NO:30 by up to three nucleotide changes, wherein the probe is capable of hybridizing to and detecting the Zika virus RNA or an amplicon thereof.   
     
     
         127 . The kit of  claim 126 , wherein the detectably labeled probe comprises a fluorophore. 
     
     
         128 . The kit of  claim 127 , wherein the detectably labeled probe comprises a 5′-fluorophore and a 3′-quencher. 
     
     
         129 - 140 . (canceled)