EP2185731A2 - Oligonukleotide, verwendung davon sowie erkennungsverfahren und kit zur feststellung der anwesenheit des e1-gens des chikungunya-virus - Google Patents

Oligonukleotide, verwendung davon sowie erkennungsverfahren und kit zur feststellung der anwesenheit des e1-gens des chikungunya-virus

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Publication number
EP2185731A2
EP2185731A2 EP08835332A EP08835332A EP2185731A2 EP 2185731 A2 EP2185731 A2 EP 2185731A2 EP 08835332 A EP08835332 A EP 08835332A EP 08835332 A EP08835332 A EP 08835332A EP 2185731 A2 EP2185731 A2 EP 2185731A2
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EP
European Patent Office
Prior art keywords
oligonucleotide
amplification
sequence
nucleotides
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP08835332A
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English (en)
French (fr)
Inventor
Jean-Noël TELLES
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Biomerieux SA
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Biomerieux SA
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Filing date
Publication date
Application filed by Biomerieux SA filed Critical Biomerieux SA
Publication of EP2185731A2 publication Critical patent/EP2185731A2/de
Ceased legal-status Critical Current

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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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Oligonucleotides use, detection method and kit for diagnosing the presence of the E1 gene of the human immunodeficiency virus
  • the present invention provides oligonucleotides for amplification and detection of a target sequence localized in the Chikungunya virus E1 gene.
  • the invention also relates to the use of these oligonucleotides, a detection method and a kit for diagnosing the presence of the El gene of Chikungunya virus.
  • the Chikungunya virus is an alphavirus arbovirus that is transmitted by Aedes mosquitoes. It was isolated for the first time in 1953. Since then, Chikungunya epidemics have occurred in tropical Africa and Asia. An unprecedented epidemic has occurred in Reunion Island, which has 775,000 inhabitants and for which more than 244,000 cases were reported as of April 20, 2006.
  • Serology is based on the detection of IgM and IgG antibodies that appear at different times during infection.
  • the IgM are detected in the serum on average 5 days after the onset of clinical signs and persist for several weeks at three month.
  • IgG are identified by two samples (acute phase and convalescence), and persist for years. The sensitivity and specificity of these tests are not well established, including the possibility of false positives by cross-reactions with IgM dengue or other arboviruses.
  • Parida et al describe the detection of Chikungunya virus by an alternative method to PCR called RT-LAMP for "reverse transcription loop-mediated isothermal amplification" (J. Clin Microbiol 2007 Feb; 45 (2): 351-7).
  • the primers were selected from the El protein gene. A sensitivity of 20 transcript copies per reaction was determined.
  • the disadvantage of this technique is that it requires 6 different primers to perform the amplification reaction.
  • the detection is not carried out with a specific probe but by turbimetry. Thus, in the case where the reaction is not specific and another virus is amplified, a false positive will be detected.
  • the present invention therefore relates to a pair of oligonucleotides for amplifying a target sequence located in the El gene of the Chikungunya virus genome, the oligonucleotide pair consisting of: a first oligonucleotide of a length between 10 and 50 nucleotides and comprising at least one fragment of 10 consecutive nucleotides derived from:
  • SEQ ID No. 1 5 '-CTCTTACCGGGTTTGTTGC-3', or its complementary sequence, and a second oligonucleotide of a length of between 10 and 50 nucleotides and comprising at least one fragment of 10 consecutive nucleotides derived from:
  • SEQ ID No. 2 5'-GCCTGGACACCTTTCGAC-3 ', or its complementary sequence.
  • the first oligonucleotide additionally comprises a promoter sequence that can be recognized by a DNA-dependent RNA polymerase enzyme.
  • the promoter sequence which can be recognized by a DNA dependent RNA polymerase enzyme is a T7 polymerase.
  • this oligonucleotide to which the promoter sequence is added, allows the amplification of the target sequence located in the E1 gene, it essentially consists of the following sequence: SEQ ID No. 3: 5'-
  • the part of the sequence in italic corresponds to the T7 promoter sequence.
  • the underlined sequence corresponds to a sequence creating a so-called linker between the promoter sequence and the oligonucleotide of sequence SEQ ID No. 1.
  • the invention also relates to an oligonucleotide to be used as a detection probe for a target sequence located in the E1 gene of the Chikungunya virus genome, the detection probe being between 10 and 50 nucleotides in length and comprising at least one fragment of 10 consecutive nucleotides derived from from:
  • SEQ ID No. 4 5'-CTCTCAGGCACCATCTGGC-S ', or its complementary sequence, said sequence comprising at least one labeling means.
  • the oligonucleotide used as a detection probe enables real-time detection and is flanked by two arms, one at the 5 'position and the other at the 3' position of said oligonucleotide, each arm having a length of between 5 and 15 nucleotides, preferably between 6 and 10 nucleotides and these arms being complementary to one another in order to present the oligonucleotide in the form of a loop in the absence of the sequence localized target in the El gene of the Chikungunya virus genome.
  • the detection probe has a fluorescent marker at the free end of one of the arms and a fluorescence absorber at the free end of the other arm.
  • the detection probe is constituted by a "molecular beacon", subsequently called molecular beacon.
  • Molecular beacons are detection probes in the form of single-stranded oligonucleotides, which have a rod-and-loop structure well known to those skilled in the art.
  • the loop contains a probe sequence complementary to the target sequence (amplicon in general), and the stem is formed by the hybridization of two sequences forming arms, which are each located at each end of the probe.
  • a fluorophore is covalently bound to the end of one of the two arms and an absorber of fluorescence (quencher) is covalently bound to the end of the other arm.
  • Molecular beacons do not fluoresce when free in solution, the structure now closes the fluorophore and absorber in close proximity to one another, resulting in the transfer of fluorescence from the fluorophore to the absorber.
  • the fluorescence absorber is a non-fluorescent chromophore that dissipates the energy received from the fluorophore in heat. However, in the presence of complementary amplicons, when they hybridize to these targets, they undergo a conformational change that allows them to fluoresce.
  • a probe-target hybrid is formed which is longer and more stable than the hybrid created by the two arms of the stem.
  • the rigidity and length of the hybrid-probe hybrid prevent the simultaneous existence of the stem hybrid.
  • the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem hybrid to dissociate and the fluorophore and absorber to move away from each other, thereby restoring fluorescence.
  • the detection probe consists of a molecular beacon preferably consisting of: SEQ ID No. 5: 5 '- [6-FAM] -CGAGCGACTCTCAGGCACCATCTGGCrCGCrCG- [DabSyl] -3', or its complementary sequence.
  • the sequence in italic corresponds to the arms mentioned above, constituting the stem.
  • the two oligonucleotides for use as amplification primers, and the oligonucleotide for use as a detection probe each have a length of between 10 and 50 nucleotides and comprise at least one fragment of 10 nucleotides consecutive sequences SEQ ID No. 2 and 4 respectively.
  • each oligonucleotide is of a length of between 12 and 30 nucleotides and comprises at least one fragment of 10 or 12 consecutive nucleotides, and even more preferably, each oligonucleotide has a length of between 15 and 26 nucleotides and comprises at least one fragment of 10, 12 or 15 nucleotides.
  • the present invention also provides the use of the pair of oligonucleotides or probe as described above, in a nucleic acid amplification reaction of the Chikungunya virus genome that may be present in a biological sample.
  • the invention further relates to a method for detecting nucleic acids of Chikungunya virus that may be present in a sample, wherein the sample is subjected to a nucleic acid amplification reaction using an oligonucleotide pair, as described above, in the presence of the amplification reagents necessary for such amplification and where the presence of amplicons of interest is detected.
  • This detection method can be based on an RT-PCR amplification reaction.
  • this detection method may be based on a transcriptional amplification technique.
  • this technique is the NASBA technique.
  • the NASBA technique is a method of isothermal amplification of nucleic acids involving several enzymatic activities, which allows rapid detection of the Chikungunya virus.
  • the amplification of nucleic acids by this route is well suited to RNA genomes, thanks to the presence of a reverse transcription step directly in the amplification reaction.
  • the invention also relates to a method method of amplifying the Chikungunya virus El gene that may be present in a sample, comprising the steps of: incubating the sample in amplification buffer in the presence of two amplification primers, each having a length of between 10 and 50 nucleotides, one additionally comprising a promoter sequence, the other of opposite polarity to the primer associated with the promoter sequence, for hybridizing respectively upstream and downstream of an area of interest located in the gene El Chikungunya virus, add the following reagents in the sample:
  • An enzyme having an RNA activity dependent DNA polymerase an enzyme having a DNA-dependent DNA polymerase activity
  • An enzyme having an RNA polymerase-dependent DNA activity and maintaining the reaction mixture thus created under appropriate conditions and for a period of time sufficient for amplification to take place.
  • the enzymes listed above are four in number but it is quite possible to use an enzyme having two or three of the above mentioned activities, in this case the use of three or two enzymes remains possible and covered. by the invention.
  • other elements are necessary to establish an amplification such as nucleotides or buffer solutions. These buffer solutions can be optimized depending on the amplification technique used or the oligonucleotides present in the reaction. In a reaction transcriptional amplification, such as a NASBA reaction, these buffer solutions may contain, for example, DMSO which enhances the amplification reaction (as described in PCT / US90 / 04733).
  • an internal control in addition, it is also possible to add to the amplification reaction an internal control, to avoid the presence of false negative due to the failure of the amplification process.
  • an internal control in a transcriptional amplification reaction is described in PCT / EP93 / 02248. Such internal control is selected such that it does not compete with the target nucleic acid in the amplification reaction. All these elements are well known to those skilled in the art.
  • the invention provides a kit for detecting the El gene of the Chikungunya virus that may be present in a sample, containing at least one pair of oligonucleotides as described above to carry out the amplification of the El gene, - at least one labeled or labeled oligonucleotide, as described above, and having a nucleic acid sequence substantially complementary to at least a portion of the amplified nucleic acid sequence, - reagents necessary for carrying out an amplification reaction .
  • the reagents necessary for carrying out an amplification reaction are reagents allowing NASBA amplification.
  • substantially complementary it is meant that hybridization is performed between a labeled or labeled oligonucleotide, otherwise known as an amplification primer. or detection probe, and at least a portion of the amplicon target or amplified nucleic acid sequence, this hybridization being specific and selective to allow amplification or detection of the amplicon of interest.
  • detecttable marker is meant at least one marker capable of directly generating a detectable signal. For example, the presence of biotin is considered direct labeling because it is detectable, although it can be subsequently associated with labeled streptavidin. A nonlimiting list of these markers follows:
  • Enzymes that produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent, luminescent compounds, colorants,
  • Electron density groups detectable by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance,
  • Detectable groups for example whose molecules are of sufficient size to induce detectable changes in their physical and / or chemical characteristics, this detection can be carried out by optical methods such as diffraction, surface plasmon resonance, surface, the variation of contact angle or physical methods such as atomic force spectroscopy, the tunnel effect,
  • radioactive molecules such as 32 P, 35 S or 125 I.
  • the marker is electrochemically detectable, and in particular the marker is a derivative of an iron complex, such as a ferrocene.
  • nucleic acid means a sequence of at least two deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, for example at least one nucleotide comprising a modified base, such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization.
  • a modified base such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base for hybridization.
  • This polynucleotide can also be modified at the level of the internucleotide linkage, for example phosphorothioates, H-phosphonates or alkylphosphonates, at the level of the backbone such as, for example, alpha-oligonucleotides (FR 2 607 507) or PNAs (M Egholm et al., J. Am Chem Soc, 114, 1895-1897, 1992 or the 2 'O-alkyl ribose and LNA (BW, Sun et al., Biochemistry, 4160-4169, 43, 2004).
  • the nucleic acid may be natural or synthetic, an oligonucleotide, a polynucleotide, a nucleic acid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA, a nucleic acid obtained by an enzymatic amplification technique such as :
  • purification step is meant in particular the separation between the nucleic acids of the microorganisms and the cellular constituents released in the lysis step which precedes the purification of the nucleic acids.
  • lysis methods such as thermal or osmotic shocks or treatments with chaotropic agents, such as guanidium salts (US-A-5, 234, 809).
  • This step generally makes it possible to concentrate the nucleic acids.
  • solid supports such as magnetic particles (see US-A-4, 672, 040 and US-A-5, 750, 338), can be used to purify nucleic acids, which are attached to these magnetic particles, by a washing step.
  • This nucleic acid purification step is particularly interesting if one wishes to subsequently amplify said nucleic acids.
  • a particularly interesting embodiment of these magnetic particles is described in patent applications WO-A-97/45202 and WO-A-99/35500.
  • the term "solid support” as used herein includes all materials to which a nucleic acid may be attached.
  • Synthetic materials or natural materials, possibly chemically modified, can be used as solid support, in particular polysaccharides, such as cellulose-based materials, for example paper, cellulose derivatives, such as cellulose acetate and nitrocellulose, or dextran; polymers, copolymers, especially based on styrene-type monomers, natural fibers such as cotton, and synthetic fibers, such as nylon; inorganic materials, such as silica, quartz, glasses, ceramics; latexes; magnetic particles; metal derivatives, gels, etc.
  • the solid support may be in the form of a microtiter plate, a membrane, a particle or a substantially planar plate of glass or silicon or derivatives.
  • the entire protocol (from the sample taken at the detection of amplicons) can be carried out in a single container or tube, processed manually or in a PLC.
  • Figures 1 to 8 Detection of Chikungunya virus at different concentrations in relative fluorescence unit (RFU ordinate) per unit of time (T on the abscissa).
  • the dotted curve corresponds to the signal emitted by the complementary probe according to the invention of a sequence of the Chikungunya and the solid line corresponds to the internal control, which is a synthetic transcript of about 1000 bases encompassing the amplified region of the NASBA primers; It will therefore be co-amplified with the wild-type sequence present in the sample.
  • the sequence indicated in bold and in italics corresponds to the T7 promoter sequence, recognized by the T7 RNA polymerase and is found in the oligonucleotides Pl for the implementation of the NASBA technique.
  • the underlined sequence corresponds to a sequence creating a gap, called a linker, between the promoter sequence and the oligonucleotide of sequence SEQ ID No. 1.
  • the doubly underlined sequences correspond to the sequences forming the rod of the molecular beacon.
  • Nucleic acids were extracted using the easyMAG System (bioMérieux BV, Boxtel, Holland) with NucliSENS Magnetic Extraction Reagents reagents (bioMérieux BV, Boxtel, Holland, NucliSENS EasyMAG Extraction Buffer 1 # 280130, NucliSENS EasyMAG Extraction Buffer 2 # 280131, NucliSENS EasyMAG Extraction Buffer 3 # 280132, NucliSENS EasyMAG Magnetic Silica # 280133, NucliSENS EasyMAG Buffer Lysis # 280134). The extractions were carried out on 200 ⁇ l of each of the plasma dilutions.
  • the dilutions correspond to concentrations of 41 copies / ml, 418 copies / ml, 4180 copies / ml, 41800 copies / ml, 418000 copies / ml, 4180000 copies / ml.
  • Results The results are visible in Figures 1 to 8 and show that the test works and detects a positive Chikungunya plasma up to the 4180 copies / ml concentration (equivalent to about 150 copies of Chikugunya RNA / NASBA reaction). It is observed that the higher the number of Chikungunya RNA copies, the greater the amplitude of the curve.
  • the results show that the internal control is correctly detected and plays its role of validating the test.
  • the internal control signal decreases in proportion to the number of Chikungunya RNA copies.
  • the signal from the internal control is at its peak in the negative plasma, and is minimal in the presence of a large number of Chikungunya RNA copies. Indeed, since it is a co-amplification, the amount of internal control has been calculated so that the internal control does not compete too much with the wild-type virus sequence.
  • the internal control is normally amplified when it is alone (negative sample), but as soon as a wild Chikungunya sequence is present, it is the amplification of this sequence which takes place until complete amplification inhibition.
  • internal control when the wild-type sequence is present in high concentration. So for the test to be validated, there must always be a signal: if the sample is negative we have positive internal control, if the sample is positive we have a positive signal with or without a signal for internal control (depending on whether the sample is strongly positive or not).
  • the primers and probe of the present invention and the reference technique were compared in NASBA and PCR, on a quantized qig of quantified chikunqunya RNA.
  • the primers / probes of the reference technique have been adapted in NASBA and those of the present invention to the reference technique.
  • a unique reaction mixture allowing the amplification and detection of Chikunqunya virus E1 E1 is prepared. Briefly, to 64 ⁇ l of diluent were added 11 ⁇ l of water, 13 ⁇ l of 1.2 M KCl, 4 ⁇ l of each of the 10 ⁇ M oliqonucletides, and 0.8 ⁇ l of the 20 ⁇ M molecular probe. A volume of 10 ⁇ l of the melanqe was then added to 5 ⁇ l of the solutions of the RNAs previously extracted. After incubation for 2 min at 65 ° C.
  • RT-PCR The one-step RT-PCR was carried out in a final volume of 20 ⁇ l containing 2.5 ⁇ l of extracted Ckikungunya RNA, and 10 ⁇ l of 2 ⁇ Thermoscript Reaction Mix Buffer (Invitrogen, Lot 1207045, Germany). , 2 pmol of Taqman probe, 9 pmol of each of the amplification primers, 25 mM MgSO4, 0.4 ⁇ l of the One Step RT-PCR enzyme mixture (Invitrogen, Lot 271375, Germany).
  • the amplifications were carried out in a LightCycler instrument (Roche, Molecular Biochemicals, Germany) using the following parameters: 50 ° C. for 20 min, 95 ° C. at 2 min and 45 cycles at 95 ° C. for 5 sec, and 60 ° C. C for 1 min.
  • R-CHIK SEQ ID NO.9: 5 '-CCAAATTGTCCYGGTCTTCCT-3'
  • F-CHIK SEQ ID NO.10: 5 '-AAGCTYCGCGTCCTTTACCAAG-3'
  • P-CHIK SEQ ID NO.11: 5 'Fam-CCAATGTCYTCMGCCTGGACACCT-Tamra
  • T and the letter M corresponds either to the base C or to the base
  • NASP1-PASTO-NAS SEQ ID NO.12:
  • F-CHIK SEQ ID NO.13: 5'-AAGCTYCGCGTCCTTTACCAAG-3 'BEAK-PASTO-NAS: SEQ ID NO.14: 5' Fam-CGAGCGACCCAATGTCYTCMGCCTGGACACCTCGCTCG-Dabsyl 3 '
  • the NASBA test is positive when the fluorescence signal obtained exceeds the negative control signal by 20%. ** The RT-PCR test is positive when a measurable Ct is obtained (the Ct is the number of cycles for which the signal becomes positive).
  • the primers and probe of the present invention can be used in RT-PCR and increase by two logarithm the sensitivity compared to the sequences of Pastorino and al. in the reference method.

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EP08835332A 2007-09-14 2008-09-11 Oligonukleotide, verwendung davon sowie erkennungsverfahren und kit zur feststellung der anwesenheit des e1-gens des chikungunya-virus Ceased EP2185731A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0757598A FR2921064A1 (fr) 2007-09-14 2007-09-14 Oligonucleotides, utilisation, methode de detection et kit permettant de diagnostiquer la presence du gene e1 du virus du chikungunya
PCT/FR2008/051625 WO2009044085A2 (fr) 2007-09-14 2008-09-11 Oligonucleotides, utilisation, methode de detection et kit permettant de diganostiquer la presence du gene e1 du virus de chikungunya

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EP2185731A2 true EP2185731A2 (de) 2010-05-19

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US (1) US20100216117A1 (de)
EP (1) EP2185731A2 (de)
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EP3957754B1 (de) 2008-04-21 2026-01-28 Gen-Probe Incorporated Verfahren zur erkennung des chikungunya-virus
WO2010071610A1 (en) * 2008-12-19 2010-06-24 Agency For Science, Technology And Research (A*Star) Severe chikungunya biomarkers
CN102414217A (zh) * 2009-02-25 2012-04-11 彼格泰格私人有限公司 用于检测基孔肯雅病毒的探针和引物
CN101935715B (zh) * 2010-09-08 2012-02-22 中国检验检疫科学研究院 实时荧光定量pcr检测基孔肯亚病毒核酸的方法
CN110607401A (zh) * 2019-10-10 2019-12-24 中国检验检疫科学研究院 一种快速检测基孔肯雅病毒的试剂盒
BR102020016662A2 (pt) * 2020-08-14 2022-03-03 Fundação Oswaldo Cruz Dispositivo portátil, método de leitura para detecção molecular automatizada de patógenos por lamp, controlado por aplicativo de smartphone e seus usos

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US20100216117A1 (en) 2010-08-26
FR2921064A1 (fr) 2009-03-20
WO2009044085A3 (fr) 2009-05-28

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