WO2012021106A1 - Détection des sérotypes 1 à 4 du virus de la dengue - Google Patents

Détection des sérotypes 1 à 4 du virus de la dengue Download PDF

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Publication number
WO2012021106A1
WO2012021106A1 PCT/SG2011/000278 SG2011000278W WO2012021106A1 WO 2012021106 A1 WO2012021106 A1 WO 2012021106A1 SG 2011000278 W SG2011000278 W SG 2011000278W WO 2012021106 A1 WO2012021106 A1 WO 2012021106A1
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seq
probe
nucleotide sequence
dengue virus
dengue
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PCT/SG2011/000278
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English (en)
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Eng Lee Tan
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Singapore Polytechnic
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Priority claimed from SG2010058287A external-priority patent/SG178629A1/en
Priority claimed from SG2011039153A external-priority patent/SG185854A1/en
Application filed by Singapore Polytechnic filed Critical Singapore Polytechnic
Priority to SG2013009253A priority Critical patent/SG187748A1/en
Publication of WO2012021106A1 publication Critical patent/WO2012021106A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to the detection of Dengue virus, and in particular the detection of individual Dengue virus serotypes. More specifically, the present invention relates to primers and probes for use in polymerase chain reaction based tests, capable of differentiating between individual serotypes of the Dengue virus. Kits of primers and probes are also provided, together with the use of the primers and probes in methods of diagnosis.
  • the Dengue virus is believed to be the most common arthropod-borne disease in the world and is transmitted to humans through the bite of a mosquito of the Aedes genus.
  • the mosquitoes require a warm climate and therefore Dengue infections are one of the most common infectious diseases in the tropical areas of the world, resulting in many fatalities over the last few decades.
  • the Dengue virus consists of 4 serotypes. They include Dengue Virus Type 1, Type 2, Type 3 and Type 4.
  • the Dengue virus has a single stranded ribonucleic (ssRNA) based genome of approximately 11000 base pairs (bp).
  • the 5' end of the RNA has a type 1 cap structure but lacks a poly A tail at the 3' end. It contains a single open reading frame of about 10,000 nucleotides encoding three structural and seven non-structural proteins.
  • the viral proteins are synthesized as a polyprotein of about 3000 amino acids. These are processed co-translationally and post-translationally by viral and host proteases.
  • the non- structural proteins are known as NS 1 to NS7.
  • NS 1 in particular is a glycoprotein that is detected in high titers in patients with secondary Dengue infections. However, its function is still unknown, although evidence have suggested that it is involved in RNA replication, and associates with the membrane on the cell surface.
  • DHF Dengue Hemorrhagic Fever
  • DSS Dengue Shock Syndrome
  • the virus again activates the immune system.
  • the immune system can be 'tricked', and may attack the virus as if it were the first serotype. This is because the 4 subtypes of Dengue have very similar surface antigens.
  • the antibodies bind to the viral surface proteins as before, but this time are unable to inactivate the virus.
  • numerous macrophages are attracted to the antibodies, but instead of removing the virus, end up becoming infected by them. This makes the viral infection much more acute.
  • the cytokines released by the body causes the endothelial tissue to become permeable, which eventually results in DHF and fluid loss from the blood vessels.
  • DSS Dengue Shock Syndrome
  • frank shock a narrow pulse pressure of 20 mm Hg, also known as frank shock. Additional complications include the liver becoming palpable and tender. In addition, liver enzymes become mildly elevated although jaundice is rare. Intense impending shock, sustained abdominal pain, persistent vomiting, restlessness or lethargy, a sudden change from fever to hypothermia with sweating and prostration are some of the symptoms that DSS is imminent. Both DHF and the more severe DSS are potentially fatal. Thus, there is a need for rapid diagnosis of Dengue infections in order for earlier clinical management to be carried out.
  • the current gold standard for Dengue virus detection and identification involves viral isolation in cell cultures or Toxorhynchites species mosquito larvae, followed by direct detection using immunofluorescence (IF).
  • IF immunofluorescence
  • Dengue diagnostics include serological diagnosis of antibodies based on the capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISA); and molecular techniques such as conventional reverse transcriptase polymerase chain reaction (RT-PCR).
  • IgM capture immunoglobulin M
  • ELISA IgG enzyme-linked immunosorbent assays
  • RT-PCR reverse transcriptase polymerase chain reaction
  • both ELISA and conventional RT-PCR take at least 6 hours to complete.
  • the IgM based assay should be repeated for at least 10 to 14 days if the initial test is negative.
  • the PCR based test requires additional post-PCR handling which increases the risks of contamination.
  • a primer or probe primer or probe having a target sequence in the conserved NS1 region of Dengue virus serotypes 1 to 4, for example, SEQ ID O:13, 14, 15 or 16.
  • a primer or probe according to claim 1 having a target sequence in one of the following regions of Dengue virus serotypes 1 to 4 :
  • a primer or probe comprising or consisting of the nucleotide sequence of any of SEQ ID NO: 1 to 12, or complements thereof.
  • a forward primer as described herein for amplifying a nucleotide sequence of a Dengue virus serotype in a test sample, in which the primer sequence comprises or consists of the nucleotide sequence of any of SEQ ID NO: 1, 4, 7 or 10, or complements thereof.
  • a reverse primer as described herein for amplifying a nucleotide sequence of a Dengue virus serotype in a test sample in which the primer sequence comprises or consists of the nucleotide sequence of any of SEQ ID NO: 2, 5, 8 or 11 , or complements thereof.
  • a set of primers for amplifying Dengue virus in a test sample comprising ' one of the following pairs of forward and reverse primers, in which the nucleotide sequences of the primers comprises or consists of the following sequences, or complements thereof:
  • a set of primers and a probe for detecting Dengue virus in a test sample comprising or consisting of the forward primers SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO: 10 or complements thereof: the reverse primers SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO: 11 or complements thereof; and the probes SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 12 or complements thereof.
  • the set of primers, comprising a forward primer and a reverse primer, and a probe, as described herein comprise or consist of the following sequences or complements thereof:
  • a method for determining the presence or absence of a Dengue virus serotype in a biological sample comprising the step of contacting a nucleotide sequence obtained or derived from the biological sample with at least one primer or probe or set as described herein.
  • the method may further comprise the step of determining whether the nucleotide sequence hybridises to the at least one primer or probe under stringent conditions, thereby detecting whether the sample contains a Dengue virus serotype.
  • in situ hybridisation is used to detect whether the nucleotide sequence hybridises to the at least one primer or probe.
  • a method as described herein comprising the steps of:
  • amplification conditions comprise an amplification reaction, in which the amplification reaction is a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • At least one forward primer comprising or consisting of a nucleotide sequence selected from the group of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10 or a complement thereof;
  • At least one reverse primer comprising or consisting of a nucleotide sequence selected from the group of SEQ ID NO:2 , SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1 or a complement thereof, under amplification conditions to generate an amplicon of a region of a dengue serotype nucleotide sequence;
  • forward primers comprising or consisting of the nucleotide sequences of SEQ ED NO: 1 , SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO: 10 or complements thereof
  • reverse primers comprising or consisting of the nucleotide sequences of SEQ ID NO:2 , SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:l 1 or complements thereof, under amplification conditions to generate amplicons of a region of dengue serotype nucleotide sequences
  • kits for detecting Dengue virus serotypes in a biological sample comprising:
  • kit as described herein, further comprising at least one probe as described herein.
  • a method of treating a patient infected with a Dengue virus comprising: determining the presence of a Dengue virus serotype in a biological sample derived from the patient using a method as described herein, and then administering an anti-viral composition or medication or a Dengue-specific
  • a composition comprising an anti-viral composition or medication or a Dengue-specific immunotherapy in the manufacture of a medicament for the treatment of a patient infected with a Dengue virus, wherein the presence of a Dengue virus serotype in a biological sample from the patient has been determined using a method as described herein.
  • a composition comprising an anti-viral composition or medication or a Dengue-specific immunotherapy for the treatment of a patient infected with a Dengue virus, wherein the presence of a Dengue virus serotype in a biological sample from the patient has been determined using a method as described herein.
  • A shows a structure of a typical TaqMan probe
  • B shows a primer and TaqMan probe binding to DNA template, Taq polymerase able to create complementary strand
  • C shows Reporter dye released from the extending double-stranded DNA created by the Taq polymerase. Once cleaved from quencher, reporter emits light.
  • Figure 4 Graph showing specific detection and differentiation of Dengue virus serotype 3 in which Detection of Dengue virus serotype 3 is at 615nm and in which other Dengue serotypes and water are present as negative control.
  • FIG. 5 Graph showing specific detection and differentiation of Dengue virus serotype 4 in which Detection of Dengue virus serotype 4 is at 670nm and in which other Dengue serotypes and water are present as negative control.
  • Figure 7 - A Gel photo after specificity test of Dengue 1 to 3 Primers with Dengue 1 to 3 RNA, in which:
  • FIG. 7 B (continued from A): Gel photo after specificity test of Dengue 4 Primers with
  • Dengue 4 RNA in which:
  • Lane 1 Primers 1 , Dengue 4 RNA
  • Figure 8 Dengue Virus Type 1 , showing conserved NS 1 sequence (SEQ ID NO: 13) and region to which the forward primer (SEQ ID NO:l) and reverse primer (SEQ ID NO:2) bind and the probe sequence (SEQ ID NO:3).
  • Figure 9 Dengue Virus Type 2, showing conserved NS1 sequence (SEQ ID NO: 14) and region to which the forward primer (SEQ ID NO:4) and reverse primer (SEQ ID NO:5) bind and the probe sequence (SEQ ID NO:6).
  • Figure 10 Dengue Virus Type 3, showing conserved NS1 sequence (SEQ ID NO: 15) and region to which the forward primer (SEQ ID NO:7) and reverse primer (SEQ ID NO:8) bind and the probe sequence (SEQ ID NO:9).
  • Figure 11 Dengue Virus Type 4, showing conserved NS1 sequence (SEQ ID NO: 16) and region to which the forward primer (SEQ ID NO: 10) and reverse primer (SEQ ID NO: 11) bind and the probe sequence (SEQ ID NO: 12).
  • Figure 12 Amplification plots of specific detection of Dengue serotype 1 to 4 in respective channels using reoptimised protocol in CFX96.
  • Figure 13 Amplification plots of specific detection of Dengue serotype 1 to 4 in respective channels using reoptimised protocol in Rotorgene.
  • Figure 14 Amplification plots of specific detection of Dengue serotype 1 to 4 in respective channels using reoptimised PCR conditions in CFX96
  • Figure 15 Amplification plots of specific detection of Dengue serotype 1 to 4 in respective channels using reoptimised PCR conditions in Rotorgene. Tables
  • SEQ ID NO: 1 Forward Primer for Dengue Virus Serotype 1 , with a target nucleotide sequence in the Dengue virus of from position 3000 to 3019.
  • SEQ ID NO:2 Reverse Primer for Dengue Virus Serotype 1, with a target nucleotide sequence in the Dengue virus of from position 3159 to 3138.
  • SEQ ID NO:3 Probe for Dengue Virus Serotype 1, with a target nucleotide sequence in the Dengue virus of from position 3035 to 3078.
  • SEQ ID NO:4 Forward Primer for Dengue Virus Serotype 2, with a target nucleotide sequence in the Dengue virus of from position 2799 to 2819
  • SEQ ID NO:5 Reverse Primer for Dengue Virus Serotype 2, with a target nucleotide sequence in the Dengue virus of from position 2956 to 2936
  • SEQ ID NO:6 Probe for Dengue Virus Serotype 2, with a target nucleotide sequence in the Dengue virus of from position 2831 to 2864
  • SEQ ID NO:7 Forward Primer for Dengue Virus Serotype 3. with a target nucleotide sequence in the Dengue virus of from position 2734 to 2752
  • SEQ ID NO:8 Reverse Primer for Dengue Virus Serotype 3, with a target nucleotide sequence in the Dengue virus of from position 2883 to 2864
  • SEQ ID NO:9 Probe for Dengue Virus Serotype 3, with a target nucleotide sequence in the Dengue virus of from position 2793 to 2819
  • SEQ ID NO: 10 Forward Primer for Dengue Virus Serotype 4, with a target nucleotide sequence in the Dengue virus of from position 2551 to 2570
  • SEQ ID NO: 1 1 Reverse Primer for Dengue Virus Serotype 4, with a target nucleotide sequence in the Dengue virus of from position 2766 to 2747
  • SEQ ⁇ ) NO: 12 Probe for Dengue Virus Serotype 4, with a target nucleotide sequence in the Dengue virus of from position 2656 to 2694
  • SEQ ID NO: 13 conserveed NS1 region for Dengue Virus Serotype 1 (from 2384bp to 3439bp of Dengue Virus serotype 1)
  • SEQ ID NO: 14 conserveed NS 1 region for Dengue Virus Serotype 2 (from 2399bp to 3454bp of Dengue Virus serotype 2)
  • SEQ ID NO: 15 conserveed NS1 region for Dengue Virus Serotype 3 (from 2393bp to 3448bp of Dengue Virus serotype 3)
  • SEQ ID NO: 16 conserveed NS1 region for Dengue Virus Serotype 4 (from 2403bp to 3458bp of Dengue Virus serotype 4)
  • nucleotide sequences presented herein are contiguous, 5' to 3' nucleotide sequences, unless otherwise described.
  • amplicon refers to a product of an amplification reaction.
  • An example of an amplicon is a DNA or an RNA product (usually a segment of a gene, DNA or RNA) produced as a result of PCR, real-time PCR, RT-PCR, competitive RT-PCR, ligase chain reaction (LCR), gap LCR, strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), or the like.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • TMA transcription-mediated amplification
  • primer is used herein to mean any single- stranded oligonucleotide-sequence capable of being used as a primer in, for example, PCR technology.
  • a “primer” refers to a single- stranded oligonucleotide sequence that is capable of acting as a point of initiation for synthesis of a primer extension product that is substantially identical to the nucleic acid strand to be copied (for a forward primer) or substantially the reverse complement of the nucleic acid strand to be copied (for a reverse primer).
  • a primer or probe sequence may be suitable for use in, for example, PCR technology. As used herein, the primer and probe sequences do not include the full length nucleotide sequence of the Dengue virus genome.
  • a primer for example its length and specific sequence, depends on the nature of the DNA and/or R A targets and on the conditions at which the primer is used, for example, temperature and ionic strength.
  • the primers may consist of the nucleotide sequences described herein, or may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more nucleotides which comprise or fall within the sequences described herein, provided they are suitable for specifically binding a target sequence, under stringent conditions.
  • the primer sequence is less than 35 nucleotides in length, for example the primer sequence is less than 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22 or 21 nucleotides in length. Slight modifications of the primers or probes, in length or in sequence, can be carried out to maintain the specificity and sensitivity required under the given circumstances.
  • probes and/ or primers described herein may be extended in length by 1, 2, 3, 4 or 5 nucleotides or reduced in length by 1, 2, 3, 4 or 5 nucleotides, for example, in either direction.
  • probe is well known in the art and is used herein to mean any single-stranded oligonucleotide sequence capable of binding nucleic acid and of being used as a probe in, for example, PCR technology.
  • Primer and probe sequences can be synthesised using any methods well known in the art.
  • nucleotide sequences can be obtained from First Base Pte Ltd, Singapore.
  • the term "comprising" in relation to the probe and/ or primer sequences described herein may be considered to include sequences that are extended in length by 1, 2, 3, 4 or 5 nucleotides, for example, in either direction.
  • any “hybridisation” is performed under stringent conditions.
  • stringent conditions means any hybridisation conditions which allow the primers to bind specifically to a nucleotide sequence within the Dengue virus serotype nucleotide sequence, but not to any other Dengue virus serotype nucleotide sequences.
  • hybridisation of a probe to a nucleic acid target region under "stringent” hybridisation conditions include conditions such as 3X SSC, 0.1% SDS, at 50°C.
  • stringent hybridisation conditions include conditions such as 3X SSC, 0.1% SDS, at 50°C.
  • the skilled person knows how to vary the parameters of temperature, probe length and salt concentration such that specific hybridisation can be achieved.
  • Hybridisation and wash conditions are well known and exemplified in, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, .Y., (1989), particularly Chapter 11 therein.
  • Specific binding or “specific hybridisation” of a probe to a region of the Dengue virus serotype nucleotide sequence means that the primer or probe forms a duplex (double- stranded nucleotide sequence) with part of this region or with the entire region under the experimental conditions used, for example under stringent hybridisation conditions, and that under those conditions the primer or probe does not form a duplex with other regions of the nucleotide sequence present in the sample to be analysed.
  • the primers and probes of the present invention that are designed for specific hybridisation within a region of the Dengue virus serotype nucleotide sequence may fall entirely within said region or may to a large extent overlap with said region (i.e. form a duplex with nucleotides outside as well as within said region).
  • target sequence is a region of the Dengue virus nucleic acid sequence (either DNA or RNA, e.g. genomic DNA, messenger R A, or amplified versions thereof) to which the sequence of the probe or primer has partial (i.e. with some degree of mismatch) or total identity; although the reverse primer is the reverse complement (or, as above, has some degree of mismatch) of the sequence it recognises.
  • the target sequence generally refers to a region of the Dengue virus sequence that differs by at least one nucleotide compared to another, or to all other, Dengue virus nucleotide sequences and is a sequence to which the primer is capable of binding under stringent conditions.
  • the target sequence is generally an "amplicon", that is, a sequence that is amplified by the primer sequences, and is a sequence to which the probe is capable of binding under stringent conditions.
  • the reverse primer fulfils these conditions to a target sequence that is the reverse complement of the primer sequence.
  • the present invention provides methods for the serotype-specific detection of Dengue virus.
  • Specific regions within non-structural protein 1 (NS1) have been identified that facilitate the specific detection of Dengue virus serotypes 1 to 4.
  • Dengue virus serotype 1 may be identified by detection of a target sequence in the region defined by SEQ ID NO: 13.
  • Dengue virus serotype 2 may be identified by detection of a target sequence in the region defined by SEQ ID NO: 14.
  • Dengue virus serotype 3 may be identified by detection of a target sequence in the region defined by SEQ ID NO: 15.
  • Dengue virus serotype 4 may be identified by detection of a target sequence in the region defined by SEQ ID NO: 16.
  • the target sequence may be identified by any means known in the art.
  • the target sequence may be detected using primer(s) and/or probe(s).
  • a forward primer and a reverse primer as described herein in which, under amplification conditions, for example in a method as described herein, the forward and reverse primer are capable of amplifying a sequence in the region from 2384bp to 3439bp for Dengue Virus type 1; 2399bp to 3454bp for Dengue Virus type 2; 2393bp to 3448bp for Dengue Virus type 3; and 2403bp to 3458bp for Dengue Virus type 4.
  • a probe as described herein that is capable of hybridising to such an amplified sequence.
  • the target sequences of the primers are in the regions identified in the column labelled "Position" in Table 1.
  • the primer or probe may be at least 95% identical to its target sequence over the length of the primer or probe, suitably greater than 95% identical such as 96%, 97%, 98%, 99% and most preferably has 100% identity over its length to the target Dengue virus serotype sequence.
  • the primers or probes of the invention may be identical to the target sequence at all nucleotide positions of the primer or probe, or may have 1, 2, or more mismatches depending upon the length of probe, temperature, reaction conditions and requirements of the assay, for example.
  • the reverse primer fulfils these conditions to a target sequence that is the reverse complement of the primer sequence.
  • each nucleotide of the primer or probe can form a hydrogen bond with its counterpart target nucleotide.
  • the complementarity of primer or probe with the target sequence is assessed by the degree of A:T and C:G base pairing, such that an adenine (A) nucleotide pairs with a thymine (T), and such that a guanine (G) nucleotide pairs with a cytosine (C), or vice versa.
  • T may be replaced by U (uracil).
  • biological sample is meant a sample of tissue or cells from a patient that has been obtained from, removed or isolated from the patient.
  • obtained or derived from is meant to be used inclusively. That is, it is intended to encompass any nucleotide sequence directly isolated from a biological sample or any nucleotide sequence derived from the sample.
  • the present invention provides a primer comprising the nucleotide sequence of any of SEQ ID NO:l, 2, 4, .5, 7, 8, 10 or 1 1, as shown in Table 1.
  • the present invention further provides a set of primers comprising one or more of the following pairs of primers :
  • the present invention further provides a probe comprising the nucleotide sequence of any of SEQ ID NO: 3, 6, 9 or 12.
  • the probes may consist of the nucleotide sequences shown in SEQ ID NO: 3, 6, 9 or 12 or may be 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18. 19 or 20, 25, 30, 35, 40, 45, 50, 75, 100 or more nucleotides which comprise or fall within the sequences of SEQ ID NO: 3, 6, 9 or 12 provided they are suitable for specifically binding a target sequence within a Dengue virus serotype nucleotide sequence.
  • the probe sequence is less than 35 nucleotides in length, for example the probe sequence is less than 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22 or 21 nucleotides in length.
  • the pair of primers should allow for the amplification of part or all of the Dengue virus serotype polynucleotide fragment to which probes are able to bind or to which the probes are immobilised on a solid support.
  • the primer and/or probe may additionally comprise a detectable label, enabling the probe to be detected. Examples of labels that may be used include: fluorescent markers or reporter dyes, for example, 6- carboxyfluorescein (6FAMTM), NEDTM (Applera
  • the detectable label is directly or indirectly attached to the probe.
  • the detectable label may comprise a fluorescent moiety attached at a 5' end of the probe.
  • the nucleotide sequence of the probe may further comprise a quencher moiety attached at a 3' end of the probe.
  • the detectable label for each of the probe sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12 is such that the probe sequences are independently detectable.
  • the probe may comprise a fluorescent reporter dye as a detectable label, as described herein, at its 5'-end and a quencher moiety at its 3 '-end.
  • the quencher may comprise a non- fluorescent quencher (NFQ).
  • a Minor Groove Binder protein (MGB; Applied Biosystems) may be added to the probe, for example the 3' end of the probe.
  • the quencher moiety may be a Black Hole Quencher (BHQ).
  • the probe may have an EcIipse(TM) Dark Quencher and an MGB(TM) moiety positioned at the 5 '-end of the probe.
  • the probe is labelled with a means of detection that differs from any other probe used in the same reaction.
  • each of the probe sequences are independently detectable, in the same reaction procedure. That is, in one embodiment, different labels, for example fluorescent dyes or markers, may be selected for differentiation between Dengue Virus types 1 to 4.
  • the labels are attached to the 5' end of the probes.
  • a quencher for example, Black Hole Quencher (BHQ), is attached at the 3' end.
  • the fluorescent dyes are selected from 6-FAM, HEX, Texas Red, and Cy5.
  • the probe for Dengue Virus type 1 is labelled with 6-FAM; the probe for Dengue Virus type 2 is labelled with HEX; the probe for Dengue Virus type 3 is labelled with Texas Red; and the probe for Dengue Virus type 4 is labelled with Cy5.
  • the sequence for each of the Dengue Virus primers and probes are shown in Table 1.
  • the TaqMan probes may be independently detectable through independent labelling with different fluorescent dyes which emit fluorescence at different wavelengths. This allows the detection and differentiation of Dengue viruses in different channels.
  • the primer and probe sequences of the present invention may contain or comprise naturally occurring nucleotide structures or bases, for example adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • modified or synthetic analogues of nucleotide structures or - bases may be included in the sequence of the probe.
  • modified or synthetic analogues of nucleotide structures or - bases may be included in the sequence of the probe.
  • modified or synthetic is meant a non-naturally occurring nucleotide structure or base.
  • Such synthetic or modified bases may replace 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the bases in the probe sequence.
  • Cytosine may be replaced by 5 -Methyl dC and Thymine may be replaced by 5-Propynyl dU.
  • a quencher such as BHQ may also be included within the sequence.
  • the present invention additionally provides a kit for detection of Dengue virus serotype 1 comprising the following components: (i) at least one primer or set of primers for Dengue virus serotype 1 as described herein; and (ii) at least one probe for Dengue virus serotype 1 as described herein.
  • the present invention further provides a kit for detection of Dengue virus serotype 2 comprising the following components: (i) at least one primer or set of primers for Dengue virus serotype 2 as described herein: and (ii) at least one probe for Dengue virus serotype 2 as described herein.
  • the present invention further provides a kit for detection of Dengue virus serotype 3 comprising the following components: (i) at least one primer or set of primers for Dengue virus serotype 3 as described herein; and (ii) at least one probe for Dengue virus serotype 3 as described herein.
  • the present invention further provides a kit for detection of Dengue virus serotype 4 comprising the following components: (i) at least one primer or set of primers for Dengue virus serotype 4 as described herein; and (ii) at least one probe for Dengue virus serotype 4 as described herein.
  • the present invention further provides a kit for detection of Dengue virus serotypes 1, 2, 3 and/or 4 comprising the following components: (i) at least one primer or set of primers for Dengue virus serotype 1, 2, 3 and 4 as described herein; and (ii) at least one probe for Dengue virus serotype 1, 2, 3 and 4 as described herein.
  • the kit comprises one forward primer, one reverse .orimer and a probe sequence which has a target sequence within the region amplified by the forward and reverse primers.
  • the set of primers are capable of amplifying a portion (amplicon) of the sequence of the Dengue virus serotype and the probe is capable of hybridising under stringent conditions to the amplicon.
  • the kit may comprise:
  • kits as described herein may further comprise amplification reagents, as required.
  • amplification reagents may include enzymes having polymerase activity, enzyme co-factors, for example magnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs), (dATP, dGTP, dCTP and dTTP).
  • the kit comprises components as shown in Table 2, Table 3 or Table 4.
  • a method for determining the presence or absence of a Dengue virus serotype in a biological sample or test sample comprising the step of contacting a nucleotide sequence obtained or derived from a biological sample with at least one primer or probe or set according as described herein.
  • the nucleotide sequence is or has been isolated from a biological sample.
  • a method of the present invention may further comprise amplifying the nucleotide sequence and detecting in the sample the amplified nucleotide sequence.
  • the method of the present invention may further comprises contacting the nucleotide sequence or amplified nucleotide sequence with one or more probes as described herein.
  • the nucleotide sequence is isolated or purified from the biological sample. In RT- PCR, genomic DNA contamination, for example additional material from a patient's sample, may lead to false positive results.
  • the genomic DNA is removed or substantially removed from the sample to be tested or included in the methods of the present invention.
  • Dengue virus serotypes may be detected using in situ hybridisation.
  • in situ hybridisation is meant is a hybridisation reaction performed using a primer or probe according to the present invention on intact chromosomes, cells or tissues isolated from a patient for direct visualization of morphologic sites of specific DNA or RNA sequences.
  • Hybridisation of the polynucleotides may be carried out using any suitable hybridisation method and detection system. Examples of hybridisation systems include conventional dot blot, Southern blots, and sandwich methods.
  • Dengue virus serotype specific nucleic acid sequences for example a probe or primer as described herein, can be labelled with biotin and the hybrid can be detected via a biotin- streptavidin coupling with a non-radioactive colour developing system.
  • a biotin- streptavidin coupling with a non-radioactive colour developing system.
  • other reverse hybridisation systems may also be employed.
  • the methods as described herein are suitable for use in a sample of fresh tissue, frozen tissue, paraffin- preserved tissue and/or ethanol preserved tissue.
  • the sample may be a biological sample.
  • biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus.
  • the sample is a whole blood sample.
  • a biological sample as contemplated herein includes cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet.
  • a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
  • a biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation.
  • nucleic acid may be used directly following extraction from the sample or, more preferably, after a polynucleotide amplification step (e.g. PCR) step.
  • a polynucleotide amplification step e.g. PCR
  • the present invention additionally provides a method of treating a patient comprising: determining whether a patient-derived biological sample tissue contains a nucleotide sequence of a Dengue virus serotype using a method as described herein, and treating the patient with a medicament or immunotherapy suitable for treatment of Dengue virus infection or the symptoms associated therewith.
  • the present invention provides a method for screening, in clinical applications, biological samples from a human patient for the presence or absence of infection by Dengue virus serotypes.
  • a method of diagnosis of a patient comprising a method or method step as described herein, in which a patient is diagnosed as suffering from or having had a Dengue virus infection of one or more Dengue virus serotypes.
  • a method of prognosis comprising a method or method step as described herein, in which a patient's prognosis is determined by whether a sample from the patient is identified as expressing more than one Dengue virus serotype, thus indicating infection from two or more Dengue virus serotypes. This may indicate an increased risk for the patient of developing Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).
  • DHF Dengue Hemorrhagic Fever
  • DFS Dengue Shock Syndrome
  • a method of treating a patient comprising determining, through use of a method of the present invention, whether the patient is infected by a Dengue virus serotype and subsequently administering a composition, medicament or immunotherapy to clear the infection or to lessen secondary symptoms or to prevent or ameliorate recurrence of disease.
  • Primers and probes of the invention may be used to detect Dengue virus serotypes using any method known in the art, non-limiting examples of which include the Polymerase Chain Reaction (PCR), for example, Real-Time Reverse Transcription (RT-)PCR.
  • PCR Polymerase Chain Reaction
  • RT- Real-Time Reverse Transcription
  • PCR is an enzyme-mediated reaction use to amplify a specific target DNA sequence. By amplifying the target DNA sequence in the DNA template, it is then able to produce millions more copies of the targeted DNA sequence. This is useful when a biological sample contains only small amounts of DNA.
  • PCR is carried out in a mixture containing DNA polymerase, a pair of primers (forward and reverse) and four deoxynucleotide triphosphates (dNTPs) with the aid of thermal cycler.
  • dNTPs deoxynucleotide triphosphates
  • primers are selected for their ability to amplify a region of a Dengue virus serotype nucleotide sequence.
  • a forward primer and a reverse primer are selected for their ability to amplify' a region of, and thus generate an amplicon of, a Dengue virus serotype 1 nucleotide sequence, a Dengue virus serotype 2 nucleotide sequence, a Dengue virus serotype 3 nucleotide sequence, or a Dengue virus serotype 4 nucleotide sequence.
  • a probe is then selected for its ability to hybridise to the sequence of the amplicon thus generated, for example, a probe is selected for its ability to hybridise to the amplicon of the Dengue virus serotype 1 nucleotide sequence, the Dengue virus serotype 2 nucleotide sequence, the Dengue virus serotype 3 nucleotide sequence, or the Dengue virus serotype 4 nucleotide sequence.
  • RT-PCR real-time reverse transcription Polymerase Chain Reaction
  • ASBA nucleic acid sequence-based amplification
  • the amplification reaction is RT-PCR.
  • the RT-PCR may be a 3-step PCR method or a 2-step PCR method.
  • the RT-PCR reaction conditions comprise a 3-step PCR method, comprising the step of thermal cycling for 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 60 seconds, for example in a CFX96 Real-Time PCR detection system.
  • the reaction mixture may be as shown in Table 2, 3 or 4.
  • the reaction mixture may be as shown in Table 2.
  • the RT-PCR reaction conditions comprise a 2-step PCR method, comprising the step of thermal cycling for 40 cycles of 95°C for 10 seconds and 60°C for 45 seconds, for example in a CFX96 Real-Time PCR detection system.
  • the reaction mixture may be as shown in Table 2, 3 or 4.
  • the reaction mixture may be as shown in Table 4.
  • the RT-PCR reaction conditions comprise a 2-step PCR method, comprising the step of thermal cycling for 40 cycles of 95°C for 10 seconds and 55°C for 45 seconds, for example in a CFX96 Real-Time PCR detection system or a Rotorgene Real-Time PCR detection " system.
  • the reaction mixture may be as shown in Table 2, 3 or 4.
  • the reaction mixture may be as shown in Table 4.
  • the methods described herein may comprise the step of synthesising cDNA from RNA at 50C for 10 minutes, followed by a reverse transcriptase deactivation at 95C for 5 minutes.
  • Real time PCR is an improved form of conventional PCR.
  • the amplicons are detected by end-point analysis, by running the DNA on agarose gel after the reaction is complete.
  • real-time PCR is able to accumulate the amplified product to be detected as the reaction is going on. This is made possible by adding to the real-time PCR reaction, a fluorescent molecule such as a DNA binding dye and probes that report the increase in the amount of DNA with a proportion increase in the fluorescent signal.
  • the probes that are commonly used are TaqMan probes.
  • the x-axis represents the PCR cycle time.
  • the amount of the PCR product is doubled in each cycle until it is enough to produce the fluorescence signal.
  • the cycle number in which this occurs is called the threshold cycle (Ct).
  • Ct threshold cycle
  • a Standard Curve is plotted to perform an absolute quantification by doing a serial dilution of the control template with known concentration.
  • the amplified standard dilution series graph would be plotted by the machine as Ct against the log of the initial copy number or concentration. Therefore the initial copy number or concentration of the unknown sample can be quantified by comparing the Ct with the help of the standard curve.
  • Figure la shows an illustrated picture of a TaqMan® probe.
  • the probe consists of two types of fluorophores: the fluorescent parts of reporter (R) dye and the quencher (Q) which is attached or unattached from the template DNA and before the polymerase acts.
  • R reporter
  • Q quencher
  • Such a probe uses fluorescence resonance energy transfer (FRET) theory.
  • the reporter dye is found on the 5' end of the probe and the quencher at the 3' end.
  • the TaqMan Probe is attached to a specific piece of template DNA after the denaturation steps. After the reaction mixture cools down, the primers anneal to the DNA.
  • Tag polymerase starts to add nucleotides and starts to cleave the TaqMan probe from the DNA template.
  • the reporter is cleaved off first. Separating from the quencher, the reporter is free to emit its energy. This is shown in Figure lc.
  • the fluorescence can be quantified.
  • NSl is a non-structural protein which codes for glycoprotein.
  • GenBank database maintained by the National Institutes of Health, USA. (http://-wyvw.ncbi.nlm.nih.gov).
  • the primers and probes were designed to ensure optimal sequence length for specificity and sensitivity.
  • the conserved NSl region is from 2384bp to 3439bp for Dengue Virus type 1; 2399bp to 3454bp for Dengue Virus type 2; 2393bp to 3448bp for Dengue Virus type 3; and 2403bp to 3458bp for Dengue Virus type 4.
  • Primers and probes were designed using Lasergene 7 software. The specificity of the primers and probes were analysed by BLAST analysis in the GenBank of the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST). A pair of primers (designated
  • DenlFP/DenlRP for Dengue type 1 (SEQ ID NO: l and 2); Den2FP/Den2RP for Dengue type 2 (SEQ ID NO:4 and 5); Den3FP/Den3RP for Dengue type 3; (SEQ ID NO:7 and 8) and Den4FP/Den4RP for Dengue type 4 (SEQ ID NO: 10 and 11).
  • TaqMan probes designated DenlProbe (SEQ ID NO:3); Den2Probe (SEQ ID NO:6); Den3Probe (SEQ ID NO:9); and Den4Probe (SEQ ID NO: 12), representing each virus type, were designed from the conserved NS 1 region of each Dengue Virus strains for specific amplification of each strain. Different fluorescent dyes were selected for differentiation between Dengue Virus types 1 to 4. The dyes were attached to the 5' end of the probes. A quencher, Black Hole
  • the fluorescent dyes selected were 6-FAM for Dengue Virus type 1, HEX for Dengue Virus type 2, Texas Red for Dengue Virus type 3 and Cy5 for Dengue Virus type 4.
  • Table 1 Nucleotide sequence of the specific primer and TaqMan probe designed for specific amplification of Dengue Virus type 1 to 4.
  • HEX hexachloro-fluorescein
  • BHQ 2 was labelled at the 3' end of Probe 3 and 4. Details of the primer and probe sequences, together with the regions of the NS 1 gene to which they bind, are given in Figures 8 to 11.
  • the multiplex real-time RT-PCR analysis was carried out using the CFX96 Real-Time PCR Detection system from Bio-Rad Laboratories, United States, with the components listed in Table 2.
  • Table 2 Components of the multiplex real-time RT-PCR setup, with adjusted volumes Reagent Volume iQ Multiplex Powermix 25.0 ⁇
  • cDNA was synthesized from RNA at 50°C for 10 minutes, followed by a reverse transcriptase deactivation at 95°C for 5 minutes. Thermal cycling was performed for 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 60 seconds in CFX96 Real-Time PCR Detection system.
  • Real-time RT-PCR analysis was carried out using the CFX96 Real-Time PCR Detection system from Bio-Rad Laboratories, United States. It uses optical technology that builds on the C I 000TM thermal cycler and with its six-channel platform combines advance optical technology, creating a precise thermal control to deliver sensitive, reliable detection that detects excitation/emission wavelengths of fluorophores ranging from 450- 730nm. Performing a real-time PCR experiment can be done with the help of the CFX Manager software.
  • the CFX96 system's solid-state optical technology consisting of six filtered LEDs, each with a corresponding filtered photodiode, maximizes fluorescence detection for specific dyes in specific channels, providing sensitive detection for precise quantification and target discrimination.
  • the optics shuttle individually illuminates and reads fluorescence from each well with high sensitivity and no cross talk. At every position and with every scan, the optics shuttle is reproducibly centred above each well, so the light path is always optimal and there is no need to sacrifice data collection in one of the channels to normalize to a passive reference.
  • the 1.2% agarose gel used in this study has a separation range of 200-5000bp in Tris- Borate-EDTA (TBE) buffer.
  • TBE Tris- Borate-EDTA
  • 2.( ⁇ L of 6X DNA Loading Dye was added to 10.( ⁇ L of amplicons.
  • 2.( ⁇ L of 6X DNA Loading Dye was added to 5.0 ⁇ of the lOObp DNA Ladder from Promega, USA.
  • the GelRedTM nucleic acid stained gel was run at voltage of 100V for 60 minutes. Bands were visualized using Molecular Imager Gel Doc XR System from Bio-Rad Laboratories, USA, after gel electrophoresis. Gel electrophoresis was conducted after every
  • the sets of primers were further tested for their specificity by running a conventional singleplex RT-PCR for detection of Dengue serotypes.
  • the PCR products were run on an agarose gel electrophoresis, as shown in Figure 7. In each reaction mix, only a single pair of primer was added together with a single RNA from each Dengue serotype.
  • the multiplex real-time RT-PCR analysis was carried out using the CFX96 Real-Time PCR Detection system from Bio-Rad Laboratories, United States, with the components listed in Table 4. The cycling conditions were changed from a 3-step PCR to a 2-step
  • cDNA was synthesized from RNA at 50°C for 10 minutes, followed by a reverse transcriptase deactivation at 95°C for 5 minutes. Thermal cycling was performed for 40 cycles of 95°C for 10 seconds and 60°C for 45 seconds in CFX96 Real-Time PCR Detection system.
  • Example 8 The conditions described in Example 8 and volumes shown in Table 4 were analysed on another Real-Time PCR Platform. The results are shown in Figure 13A, 13B, 13C and 13D. It was observed that the signals were very low and unstable, as indicated by the staggering fluorescence signals and the dipping of the signals near the end of the PCR cycles.
  • PCR conditions were further fine-tuned to evaluate their specificity and stable fluorescence signalling on both the CFX96 and Rotorgene real-time PCR platforms.
  • the volumes of reagents were the same as those used in Table 4.
  • the 2-step PCR conditions were amended as shown in bold, below.
  • cDNA was synthesized from RNA at 50°C for 10 minutes, followed by a reverse transcriptase deactivation at 95°C for 5 minutes. Thermal cycling was performed for 40 cycles of 95°C for 10 seconds and 55°C for 45 seconds Similar conditions were applied to both CFX96 and Rotorgene real-time PCR platforms. The results are shown in Figures 14A, 14B, 14C and 14D, and 15A, 15B, 15C and 15D.
  • Real time RT-PCR has greatly reduced the time needed for an assay compared to conventional PCR. It reduces the experiment time by at least 5 hours to a day.
  • This method requires skill to be done as the probe and primer is costly to be subjected to any wastage.
  • This assay also reduce the amount of sample needed, thus low viral load can be tested. This is a great advantage when sample available is limited. Also, the test is done in a close-tube system when reduce the level of contamination to the lowest.
  • this method is best suited for clinical evaluation as it has the high throughput, sensitivity and specificity in the assay that are required when it comes to large breakouts of Dengue fever.
  • the assay described herein targets the non-structural protein NSl, which codes for a glycoprotein.
  • NSl protein was specifically chosen as a target for the present assay.
  • Capsid proteins and other proteins have been selected in other assays, NS 1 is involved in viral replication and appears to be the most stable protein in Dengue virus serotypes.
  • the present assay may still specifically and selectively detect future mutated Dengue virus serotypes.
  • the primers and probes do not form any secondary structure formations and are effective in rapidly differentiating between the four different Dengue virus serotypes.

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Abstract

La présente invention concerne des amorces et des sondes ayant une séquence cible dans la région NS1 conservée des sérotypes 1 à 4 du virus de la dengue.
PCT/SG2011/000278 2010-08-10 2011-08-10 Détection des sérotypes 1 à 4 du virus de la dengue WO2012021106A1 (fr)

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CN104630388A (zh) * 2014-09-10 2015-05-20 广州市第八人民医院 一种登革病毒快速分型鉴别检测试剂盒
KR101541957B1 (ko) 2013-06-25 2015-08-05 원광대학교산학협력단 ４가지 혈청형 뎅기 바이러스의 다중 동시 감별 진단용 올리고뉴클레오티드 및 이의 용도
CN105441588A (zh) * 2015-12-21 2016-03-30 深圳市生科源技术有限公司 登革热ⅰ、ⅱ、ⅲ、ⅳ型rt-pcr一步mix检测试剂盒及其检测方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101541957B1 (ko) 2013-06-25 2015-08-05 원광대학교산학협력단 ４가지 혈청형 뎅기 바이러스의 다중 동시 감별 진단용 올리고뉴클레오티드 및 이의 용도
CN104630388A (zh) * 2014-09-10 2015-05-20 广州市第八人民医院 一种登革病毒快速分型鉴别检测试剂盒
CN105441588A (zh) * 2015-12-21 2016-03-30 深圳市生科源技术有限公司 登革热ⅰ、ⅱ、ⅲ、ⅳ型rt-pcr一步mix检测试剂盒及其检测方法
CN105441588B (zh) * 2015-12-21 2019-01-15 深圳生科原生物股份有限公司 登革热ⅰ、ⅱ、ⅲ、ⅳ型rt-pcr一步mix检测试剂盒及其检测方法

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