Methods and reagents for detection, quantitation, and serotyping of dengue viruses
View Patent ↗Methods and oligonucleotide reagents for diagnosing dengue virus infection are described. In particular, the invention relates to methods for detection, quantitation, and serotyping dengue virus, including serotypes 1-4. The dengue virus can be specifically detected by these methods even in samples containing other viruses, such as West Nile virus, Japanese encephalitis virus, tick-born encephalitis virus, HIV, or HCV.
1. A method for detecting dengue virus in a biological sample, the method comprising:
(a) contacting a biological sample suspected of containing dengue virus with a set of primers comprising:
(i) a forward primer comprising the sequence of SEQ ID NO:5 and a reverse primer comprising the sequence of SEQ ID NO:9;
(ii) a forward primer comprising the sequence of SEQ ID NO:5 and a reverse primer comprising the sequence of SEQ ID NO:10;
(iii) a forward primer comprising the sequence of SEQ ID NO:5 and a reverse primer comprising the sequence of SEQ ID NO:11;
(iv) a forward primer comprising the sequence of SEQ ID NO:6 and a reverse primer comprising the sequence of SEQ ID NO:10;
(v) a forward primer comprising the sequence of SEQ ID NO:6 and a reverse primer comprising the sequence of SEQ ID NO:11;
(vi) a forward primer comprising the sequence of SEQ ID NO:7 and a reverse primer comprising the sequence of SEQ ID NO:9;
(vii) a forward primer comprising the sequence of SEQ ID NO:8 and a reverse primer comprising the sequence of SEQ ID NO:12;
(viii) a forward primer and a reverse primer each comprising at least 10 contiguous nucleotides from the corresponding nucleotide sequences of the forward primer and reverse primer of a primer set selected from the group consisting essentially of (i)-(vii);
(ix) 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 a primer set selected from the group consisting essentially of (i)-(vii) 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 dengue virus nucleic acids in the nucleic acid amplification assay;
(x) 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 essentially of (i)-(ix); or
(xi) a combination of a primer set selected from the group comprising (i)-(x),
(b) amplifying dengue virus nucleic acids if present, said nucleic acids comprising at least one target sequence selected from the group consisting essentially of a dengue-1 target sequence, a dengue-2 target sequence, a dengue-3 target sequence, and a dengue-4 target sequence;
(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 dengue virus RNA or amplicon thereof, if present, as an indication of the presence or absence of dengue virus in the sample.
2. The method of claim 1 , wherein at least one probe is selected from the group consisting essentially of:
(a) a probe comprising the sequence of SEQ ID NO:13,
(b) a probe comprising the sequence of SEQ ID NO:14,
(c) a probe comprising the sequence of SEQ ID NO:15,
(d) a probe comprising the sequence of SEQ ID NO:16,
(e) a probe comprising the sequence of SEQ ID NO:17,
(f) a probe comprising the sequence of SEQ ID NO:18,
(g) a probe comprising the sequence of SEQ ID NO:19,
(h) a probe comprising the sequence of SEQ ID NO:20,
(i) a probe comprising the sequence of SEQ ID NO:21,
(j) a probe that differs from the corresponding nucleotide sequence of a probe selected from the group consisting essentially of (a)-(i) in that the probe has up to three nucleotide changes compared to the corresponding sequence, wherein the probe is capable of hybridizing to and detecting the dengue virus RNA or amplicon thereof, and
(k) a combination of probes selected from the group comprising (a)-(j).
3. The method of claim 1 , wherein a set of probes is used for detecting dengue virus in a biological sample, wherein the set of probes comprises a probe comprising the sequence of SEQ ID NO:13, a probe comprising the sequence of SEQ ID NO:14, a probe comprising the sequence of SEQ ID NO:15, and a probe comprising the sequence of SEQ ID NO:16.
4. The method of claim 1 , wherein the set of primers used for detecting dengue virus in the biological sample comprise a primer comprising the sequence of SEQ ID NO:5, a primer comprising the sequence of SEQ ID NO:6, a primer comprising the sequence of SEQ ID NO:7, a primer comprising the sequence of SEQ ID NO:8, a primer comprising the sequence of SEQ ID NO:9, a primer comprising the sequence of SEQ ID NO:10, a primer comprising the sequence of SEQ ID NO:11, and a primer comprising the sequence of SEQ ID NO:12.
5. The method of claim 1 , wherein the set of primers used for detecting dengue virus in the biological sample comprise
(a) a primer comprising the sequence of SEQ ID NO:5,
(b) a primer comprising the sequence of SEQ ID NO:8,
(c) a primer comprising the sequence of SEQ ID NO:9,
(d) a primer comprising the sequence of SEQ ID NO:12, and
(e) a primer comprising the sequence of SEQ ID NO:10, a primer comprising the sequence of SEQ ID NO:11, or a primer comprising the sequence of SEQ ID NO:10 and a primer comprising the sequence of SEQ ID NO:11.
6. The method of claim 4 , wherein the set of primers used for detecting dengue virus in the biological sample further comprise a primer comprising the sequence of SEQ ID NO:6 or a primer comprising the sequence of SEQ ID NO:7.
7. The method of claim 1 , wherein amplifying comprises reverse transcriptase polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), or a fluorogenic 5′ nuclease assay, or a combination thereof.
8. The method of claim 2 , wherein the primers and probes that are used for detecting dengue virus in a biological sample comprise a primer comprising the sequence of SEQ ID NO:5, a primer comprising the sequence of SEQ ID NO:6, a primer comprising the sequence of SEQ ID NO:7, a primer comprising the sequence of SEQ ID NO:8, a primer comprising the sequence of SEQ ID NO:9, a primer comprising the sequence of SEQ ID NO:10, a primer comprising the sequence of SEQ ID NO:11, a primer comprising the sequence of SEQ ID NO:12, a probe comprising the sequence of SEQ ID NO:13, a probe comprising the sequence of SEQ ID NO:14, a probe comprising the sequence of SEQ ID NO:15, and a probe comprising the sequence of SEQ ID NO:16.
9. The method of claim 1 , further comprising serotyping the dengue virus present in the biological sample by detecting the presence of at least one target sequence selected from the group consisting essentially of a dengue-1 target sequence, a dengue-2 target sequence, a dengue-3 target sequence, and a dengue-4 target sequence.