WO2012153153A1 - Procédé de détermination rapide de virus à l'aide de diagnostics moléculaires basés sur des acides nucléiques, et trousse pour sa mise en œuvre - Google Patents

Procédé de détermination rapide de virus à l'aide de diagnostics moléculaires basés sur des acides nucléiques, et trousse pour sa mise en œuvre Download PDF

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WO2012153153A1
WO2012153153A1 PCT/HU2011/000045 HU2011000045W WO2012153153A1 WO 2012153153 A1 WO2012153153 A1 WO 2012153153A1 HU 2011000045 W HU2011000045 W HU 2011000045W WO 2012153153 A1 WO2012153153 A1 WO 2012153153A1
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seq
procedure
sequences
virus
favourably
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Gábor KISS
János KISS
Timea Kiss
Dániel Péter PAPP
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Diagon Kft.
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    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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    • C12Q1/708—Specific hybridization probes for papilloma
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • 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
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, in the course of which qualitative and quantitative determination of DNA viruses or RNA viruses in samples is performed using multiplex realtime PCR technique in such a way that in changeable genetic surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously with the help of flexible oligonucleotide probes sensitive for spatial conformation planned by us. Furthermore, the specification relates to a KIT for the practical realisation of the procedure.
  • the etiological screening and periodical testing of certain human diseases of viral origin is essential both in respect of public health and clinical medicine, as for example the presence of pathogenic viruses represents an increased risk not only to host organisms carrying the virus, but also to healthy persons potentially targeted by communal, therapeutic, environmental vectors transmitting viral pathogens.
  • An example of being targeted by the latter type of vectors is the direct use of virus-infected (hepatitis, influenza, etc.) environmental waters or animal and plant-based nutrient sources, or the inadequacy of the hygienic conditions of certain medical interventions, or viral infections spread through insect bites (influenza, dengue fever, etc.).
  • the abbreviation DNA is used for deoxyribonucleic acid, which can be double-stranded or single-stranded in structure.
  • the abbreviation RNA is used for ribonucleic acid, which can be partly double-stranded or single-stranded in structure.
  • viral genome relates to the entire genetic material carried by viral DNA or viral RNA, all nucleotide sequences.
  • the term viral genotype, genotype variant relates to all taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences of the given viral genome variant.
  • RT reverse transcription means the transcription of the initial RNA as template sequence into cDNA complementary DNA sequence using RNA-dependent DNA polymerase reverse transcriptase enzyme.
  • PCR method is used to refer to the in vitro enzymatic amplification of single-stranded DNA chains, the widely known technique of polymerase chain reaction.
  • RT reverse transcription and PCR are combined in the RT-PCR reverse transcription PCR technique, which, in the present procedure, is realised using the faster single-step, same-space RT-PCR reverse transcription PCR technique, which involves a lower risk of contamination.
  • the real-time PCR method is used to relate to a version of the above PCR and RT-PCR reverse transcription PCR nucleic acid amplification, in which nucleic acid amplification during the reaction is monitored with the help of PCR probe element (see below) fluorescent-labelled oligonucleotides, so-called internal probes, as a result of which at the end of the real-time PCR process the reaction kinetics can be summarised.
  • PCR probe element see below
  • fluorescent-labelled oligonucleotides so-called internal probes
  • PCR probe element(s) relates to short, single-stranded oligonucleotide DNA sequences delimiting, directing and determining the selective specific detectability of PCR amplification of single-stranded DNA chains, including: forward primer(s), reverse primer(s), primer pair(s) consisting of the two former ones together, and probe(s) labelled for the purpose of detection.
  • hybridisation annealing is used to refer to the complex of complementary nucleotide sequences formed with secondary chemical bonds, hydrogen bonds, that is their hybridisation pairing.
  • thermodynamically strong hybridisation refers to the hybridisation pairing of complementary natural nucleotides, nucleotide sequences.
  • thermodynamically weak hybridisation refers to the hybridisation pairing of degenerate and semi-degenerate nucleotides, nucleotide sequences.
  • genetic code "wobble" is the biological phenomenon when in the genomic polynucleotide DNA or RNA chain consecutive trinucleotides, nucleotide base triplets built from A adenine, T thymine, G guanine, C cytosine or U uracil nucleotides provide the different genetic signals, genetic codes determining diverse amino acids.
  • the messenger RNA nucleotide triplet codon complementary to the code is complementary to the transfer RNA anticodon nucleotide triplet sequence carrying the protein-building amino acid, that is it establishes hybridisation pairing with it.
  • An amino acid can be determined by several different nucleotide triplet sequences, consequently an amino acid can have several different codons.
  • degenerate sequence is used to refer to the nucleotide or oligonucleotide sequence, which, as the natural feature of the genetic code, is characterised by that the same amino acid can be coded by several different base triplets, that is nucleotide triplets.
  • degenerate oligonucleotide is used to refer to an oligonucleotide, in the sequence of which any of the A adenine, T thymine, G guanine, C cytosine or U uracil nucleotide bases can occur in the individual positions.
  • degenerate oligonucleotide also relates to a synthetic nucleotide sequence, where any type of nucleotide can occur in any nucleotide position.
  • any of the A adenine, T thymine, G guanine, C cytosine nucleotides can occur in the "n" positions of nucleotide sequences.
  • semi-degenerate oligonucleotide is used to refer to a synthetic nucleotide sequence, in which degenerations occur only in the given nucleotide positions and according to given rules. In the present specifications there are the following rules of semi-degeneration. Base position Possible nucleotides
  • palindromic sequences are nucleotide sequences, which, when read from the two ends of a single-stranded polynucleotide, oligonucleotide chain, show the same nucleotide sequence.
  • a standard testing possibility in the indirect detection of the presence of viruses is immunoserology, when infection is indicated by the in vitro detection of immunoglobulins, antibodies specific for surface and internal antigens of the virus, produced by the infected organism.
  • Sensitive and specific immunoserologic methods concentrate mainly on the detection of antibodies specific to virus envelope surface membrane antigen determinants, nucleocapsid core antigen determinants below them, and viral genome antigen determinants embedded in nucleoprotein within the nucleocapsid core, that is on the detection of serum IgM and IgG immunoglobulins specific to viral antigen determinants listed above.
  • Such immunoserologic methods include for example HI hemagglutination inhibition, CF complement fixation, NA detection of the presence of neutralising antibodies, IF immunofluorescence detection, RIA radioimmunoassay or EIA enzyme-linked immunoassay [see: Wong's Virology: Diagnostic Methods in Virology http://virology- online.com/general/Tests.htm].
  • the above immunoserologic methods indirectly prove viral infection, such as for example in patent specification no. US 7595152, where the presence of the Influenza A virus and the Influenza B virus is proved by detecting antibodies specific to virus NS1 non-structural protein domain 1 epitopes.
  • Another type of indirect detection is the immunodiagnostic antibody determination for the presence of the Hepatitis A virus described in the summarising work by Nainan O.V. et al. [Nainan O.V. et al (2006): Clin. Microbiol. Rev. 19: 63-79.].
  • a further example of the indirect immunoserologic demonstration of virus infection is the simultaneous detection of Hepatitis B and Hepatitis C virus specific antibodies with the help of a protein chip assay using nano-gold immuno- amplification [Duan L. et al.(2005): BMC Infectious Diseases 5:53, http://www.biomedcentral.eom/1471-2334/5/53]. Bichr S. et al.
  • a classic standard testing possibility for the direct demonstration of the presence of viruses is the in vitro detection of the virus particles using qualitative (that is morphologic- immunologic typing of virus) and quantitative (that is virus number, virus titre determination) methods in blood serum, tissues and cells, and in the culture media and cells of the in vitro tissue culture experimentally infected with the virus [Roingeard P. (2008): Biol. Cell 100: 491-501.].
  • the direct detection of the presence of pathogenic viruses as above may be significantly influenced by the viral replication rate, the viral titre in blood serum, and the different cytopathogenic (that is leading to degenerative changes) nature of the virus in the infected target tissues.
  • nucleic acid-based molecular diagnostic methods were introduced, such as nucleic acid amplification diagnostic tests using the widely known traditional PCR method [see patent specifications no. US 4683195, US 4683202, US 4889818, US 4965188].
  • the advantage of these diagnostic tests is that even in the case of a low virus numbers they are operating standard applications recognised all over the world, and in respect of their demand of chemicals, instruments and infrastructure they can be performed with the automated instrumental system of a clinical laboratory.
  • the disadvantage of such diagnostic tests is their time-consuming nature, their demand for high-value instruments and their dependence on numerous external effects or user faults while preparing reactions.
  • endpoint assay is difficult in the case of the traditional nucleic acid amplification PCR or RT-PCR reverse transcription PCR method, or the optimisation of separation of products according to size by gel electrophoresis, as human factors have a significant influence on the result in final evaluation.
  • the monoplex real-time PCR and the monoplex single-step real-time RT-PCR methods enable the examination of a template to be detected in a given sample, and through this the examination of a type of PCR product amplicon
  • multichannel real-time PCR instruments make it possible to obtain information about several templates to be detected in a testing system.
  • several detection reactions are performed and by using different fluorescent labels, the template sequences to be determined are detected according to the wavelengths of the different fluorescent channels. Consequently, in the same sample, in one step, in one reaction space several templates to be detected and several types of PCR product amplicons can be examined, as a result of which the reliability of the diagnostic result can be enhanced.
  • the hit accuracy of the diagnostic methods is significantly influenced by the relatively high genetic changeability of the viruses, which may be manifested for example in the different expression of a surface antigen determinant, or for example in the altered hybridisation annealing of the PCR probe elements - indicating hits in nucleic acid amplification diagnostic tests - to variable genetic target sequences. This is why we find it necessary to plan marker PCR probe elements, the hybridisation annealing of which, under the biochemical and physical circumstances of the diagnostic tests,
  • ⁇ proves the presence and titre of the virus with a taxonomically reliable diagnosis.
  • thermodynamic stability depends on the mutations in template sequence to be detected characteristic of the genotype of given viral strain, which makes it necessary to design sensitive PCR probe elements for specific diagnostic determination.
  • the cause of this phenomenon is the high genetic changeability of viruses, as the errors of high rate nucleic acid replication supporting rapid multiplication, the distortion or lack of repairing-correcting enzymatic processes, and the characteristically small size of the viral genome all provide a favourable background for the fairly rapid accumulation of different types of mutations [Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P.: Molecular Biology of the Cell, 5 th edition, Garland Science, 2008; Murray P.R., Rosenthal K.S., Pfaller M.A.: Medical microbiology, 6 th edition, Mosby, Elsevier, 2009].
  • a diagnostically significant consequence of the high genetic changeability of viruses is that in the case of the same infectious viral strain, among the mature virions released from the infected host cell often there are genetic variants, in the detection of which PCR probe elements do not anneal to the right template positions, and this changed selectivity leads to a false positive result. At the same time a false negative result is obtained, if in the test sample, despite the presence of the viral nucleic acid to be detected, the specific hybridisation annealing of the PCR probe elements does not take place.
  • oligonucleotide probes e.g. Molecular Beacon, Scorpion
  • the closed condition of these spatial stem-loop structured single-stranded oligonucleotide probes is ensured by the formation of the stem, that is by the template- independent semi-degenerate sequences of the two ends of the nucleotide chain hybridising to each other.
  • the single internal loop held together by the stem contains the nucleotide sequence complementary to the template sequence to be detected.
  • the less stable stem opens out, as a result of which at the two ends of the oligonucleotide chain the fluorescent donor and acceptor-quenching labels move away from each other in space, the emission and absorption wavelength spectra stop overlapping each other, and so the reporting fluorescent signal can be detected [see US Patent no. 6174670 on donor and acceptor oligonucleotide probes, and US Patent no. 5538848 on quenching fluorescence probes].
  • thermostable DNA polymerase catalysing chain extension in the PCR reaction [Broude N.E.(2005): Encyclopedia of Diagnostic Genomics and Proteomics DOI: 10.1081 E-EDGP 120020717].
  • oligonucleotide probe for PCR-based diagnostic determination of the presence of viral nucleic acid, PCR probe elements containing degenerate - semi- degenerate sequences are needed, which indicate the hits of our PCR reactions reliably and selectively in changeable sequence surroundings.
  • oligonucleotide probe A special feature of our oligonucleotide probe is that in the single-stranded chain planned by us palindromic motifs are created, and multiple internal loops are formed by thermodynamically weak hybridisation pairing of semi-degenerate bases placed in them (figure 3). With these internal loops the structural flexibility of the probe is ensured, as a result of which variable sequences can be bound with our probe.
  • variable sequences can be founded by providing the close proximity of the multiple internal loops in our oligonucleotide probe with nucleotide sequences like bridge abutments, and with the latter nucleotide sequences thermodynamically strong hybridisation is realised on stable nucleotide sequences of the template to be detected.
  • This strong hybridisation annealing opens up the semi-degenerate internal loops, as a result of which, by flexibly spanning the variable regions around the stable template sequences, our probe can be bound to variable sequences.
  • a further characteristic feature of our oligonucleotide probe with multiple internal loops is that on the template to be detected the sequence of a length of at least 25-30 nucleotides suiting our expectations can be bound in a thermodynamically stable and selective way. Due to the multiple internal loops and the structural flexibility described above, we gave the oligonucleotide probe planned by us the attributives sensitive for spatial conformation and flexible, and we named it Camel probe (figure 3).
  • taxonomically characteristic stable consensus and subtype characteristic unique variable sequences are detected simultaneously with the help of flexible oligonucleotide probes sensitive for spatial conformation planned by us, the fluorescence detection of which is favourably solved with complementary strand displacement hydrolysis described above (figure 1, figure 3).
  • the target template sequences are detected reliably in changeable sequence surroundings.
  • buffer medium such as the 5x MasterMix buffer medium according to table 1 for the determination of Influenza A virus according to our procedure, or for example the 5x MasterMix buffer medium according to table 4 for the determination of Hepatitis C virus according to our procedure
  • the target template sequences are detected reliably in changeable sequence surroundings.
  • the genotype variations of given virus mutations are detected reliably because the changeable sequences surrounding template stable positions to be detected fit appropriately in our flexible probe sensitive for spatial conformation.
  • Influenza A virus Orthomyxoviridae / Influenzaviridae
  • this name is used to refer to the etiological factors of seasonal influenza.
  • the structure of the Influenza A virion we can distinguish the envelope, below it the M matrix region rich in protein, below it the internal helical nucleocapsid core assembly with the negative-strand RNA genome inside it embedded in NP nucleoprotein and arranged in eight segments.
  • the M matrix protein and the NP nucleoprotein are immunological determinants, which antigens characteristically distinguish the entities of the Influenza A genus from the entities of the Influenza B and Influenza C genus.
  • the immunoserological detection of the Influenza A viral strains is based on the characteristic unique combination of the HA haemagglutinin lectin and the NA neuraminidase enzyme glycoprotein antigens situated in the envelope of the virion, playing a role in the lifecycle and pathogenicity of the virus, specific to the given viral strain.
  • Influenza A positivity is used to refer to the presence of Ml matrix protein 1 and M2 matrix protein 2 coding sequences.
  • the variable regions coding the HA haemagglutinin lectin, the NA neuraminidase enzyme and the NP nucleoproteins are suitable for the genotype level classification of the Influenza A virus serotype combinations, such as: Orthomyxoviridae / Influenzaviridae / Influenza A virus / H1N1 variant / swine zoonosis origin, marked by us as AHlNlswl.
  • mainly two-step endpoint measurement RT-PCR procedures thermocycling + electrophoresis
  • the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples.
  • Deneue virus (Flaviviridae / Flavivirus) - this name is used to refer to the etiological factors of dengue fever, dengue haemorrhagic fever, dengue shock syndrome, the DV1, DV2, DV3, DV4 type viruses that are serologically related but have different immunodeterminants.
  • the positive-stranded single-segment RNA genome of the Dengue virion codes several non-structural proteins and specific serologically dominant structural proteins of the viral envelope, the delimiting membrane below it and the internal nucleocapsid core.
  • the Dengue positivity relates to the presence of the CP capsid polyprotein coding sequences.
  • the genotype classification of four serotypes of the virus can be determined, such as: Dengue virus / DV1, DV2, DV3, DV4 genotypes.
  • RT-PCR procedures thermocycling + electrophoresis
  • nested PCR or the Sanger sequencing method is used for genotyping.
  • sensitive and specific instrumental molecular diagnostic procedure can be used for the fast preliminary screening of samples raising the suspicion of Dengue infection, on the 1 st or 2 nd day following infection.
  • Hepatitis A virus (Picornaviridae / Hepatovirus) - this name is used to refer to viruses causing infectious hepatitis, jaundice, and showing the same serotype in biogeographic distribution.
  • Hepatitis A virus virion which does not have an envelope, within the nucleocapsid core there is the single-segment positive- stranded RNA genome, which contains, for example, sequences coding the three main structural proteins of the nucleocapsid core (VP-1, VP-2, VP-3) and several non-structural proteins.
  • the Hepatitis A virus positivity relates to the presence of genetically stable region of the viral genome, gene region VP-1 coding the HsAg hepatitis A virus surface antigen determinant.
  • the genotype can be described with hsag-HA V variable sequences.
  • Hepatitis A virus positive samples indicating the same serotype there is no further genetic differentiation.
  • two-step endpoint measurement RT-PCR procedure thermocycling + electrophoresis
  • PCR colorimetry Cobas Amplicor
  • Hepatitis B virus Hepadnaviridae
  • this name is used to refer to the etiological factors of viral hepatitis causing chronic liver disease progressing to liver cirrhosis or hepatocellular carcinoma, the hepatotropic viruses having different serotypes and genotypes in biogeographical distribution.
  • the structurally entire viral particle, the Hepatitis B virus virion is covered by the viral envelope, below which the internal nucleocapsid core carries the single-stranded circular DNA genome.
  • the Hepatitis B virus positivity relates to the presence of the genetically stable c gene region coding the structural constituent of the virion's nucleocapsid core.
  • Hepatitis C virus (Flaviviridae) - this name is used to refer to the etiological factors of the so-called non-A non-B hepatitis, and cirrhosis or hepatocellular carcinoma developing from it. Having a varying genotype according to its biogeographic distribution, the entire virus particle is covered with the viral envelope, below which the internal nucleocapsid core contains the positive- stranded single-segment RNA genome.
  • the envelope proteins, core proteins and NSP non-structural proteins supporting virus adsorption and penetration and specific for Hepatitis C virus are all proteolytic cleavage products of a common polyprotein.
  • the Hepatitis C virus positivity of the sample relates to the presence 5' UTR untranscribing non-coding stable genetic region of the viral genome.
  • Human immunodeficiency virus (Lentiviridae) - this name is used to refer mostly to retroviruses damaging CD4+ T lymphocytes, monocytes, macrophages, dendritic cells and neurocytes.
  • the nucleocapsid core containing p24 protein covers the positive-stranded two-segment RNA genome.
  • the specific structural constituents of the envelope are virus adsorption and penetration supporting glycoproteins (e.g. gpl20, gp41) and the matrix between the envelope and the nucleocapsid core, rich in protein.
  • the Human immunodeficiency virus positivity of the sample relates to the presence of nef envelope protein coding sequences.
  • variable sequences inside the nucleocapsid coding gag (gag I, II) gene are suitable for the genotype level classification of serotype I and II of the virus, such as: Human immunodeficiency virus HIV-I, HIV-II serotype.
  • two-step endpoint measurement RT-PCR procedures thermocycling + electrophoresis
  • PCR colorimetry Cobas Amplicor
  • Human papillomavirus (Papillomaviridae) - this name is used to refer to pathogenic viruses responsible for different malformations of epithelial origin. Presently more than 130 different types of Human papillomaviruses can be distinguished; their great diversity is due to the accumulation of frequent genetic mutations accompanying the increased propensity to multiply specific for viruses. In Human papillomavirus, not having a viral envelope, inside the nucleocapsid core there is the double-stranded DNA genome, which also codes early and late proteins ensuring settlement and multiplication in the host cell.
  • HPV-L and so-called high-risk (HPV-H) infections can be distinguished depending on whether the current infection is caused by the viral genotype inducing milder (for example warts) or more severe (for example tumours of epithelial origin) phenotype alterations.
  • the Human papillomavirus positivity of the sample relates to the presence of HPV-L genotype variant E6 early protein coding e6 sequences.
  • the viral genotype can be differentiated with unique variable proto-oncogene nucleotide sequences coding proteins of Ll-L low-risk factors and proteins of Ll-H high-risk factors.
  • the subject of the invention is a procedure for the rapid determination of the above viruses, and other DNA viruses or RNA viruses by extending our scope of protection, using nucleic acid-based molecular diagnostics, in the course of which qualitative and quantitative determination of DNA viruses or RNA viruses in samples is performed using multiplex realtime PCR technique in such a way that in changeable genetic surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously with the help of flexible oligonucleotide probes sensitive for spatial conformation planned by us.
  • multiplex multicolour real-time PCR technique is used for the simultaneous determination of several templates to be detected.
  • a buffer medium is set up, an example of which is the 5x MasterMix buffer medium for the determination of Influenza A virus shown in table 1 (also see implementation example 1), or the 5x MasterMix buffer medium for the determination of Hepatitis C virus shown in table 4 (also see implementation example 2).
  • degenerate - semi-degenerate annealing positions are established in the PCR probe element oligonucleotides, as it can be observed for example in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 oligonucleotides designed for determining Influenza A virus and in SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50 oligonucleotides designed for determining Hepatitis C virus.
  • the nucleotide sequences in the bridge abutment-like close proximity of the multiple internal loops are planned in such a way that they are captured on the stable nucleotide sequences of the template to be detected, accessible in international genetic databases, via thermodynamically strong hybridisation.
  • This strong hybridisation annealing opens up the internal loops, as a result of which, by flexibly spanning the variable regions around the stable template sequences to be detected, our probe can be bound to variable sequences.
  • An example of our flexible oligonucleotide probe sensitive for spatial conformation as above is the SEQ ID NO 50 oligonucleotide probe, designed for determining Hepatitis C virus la/lb genotypes.
  • the unique variable regions specific for the given viral genome subtype or genotype are favourably selected in accordance with the international professional protocols, by studying the international bioinformatics databases [for example www.ncbi.nlm.nih.gov, www.embl.org, www.jdb.com].
  • the probe designed by us contains multiple internal loops with semi- degenerate bases suiting the variable regions of the virus to be currently detected, favourably suiting the selected variable sequences expressed in numbers in the international professional protocols.
  • the multiplex multicolour detection of the several types of PCR product amplicons is solved by attaching different fluorescent labels to the PCR probe element oligonucleotide probes planned by us, and by complementary strand displacement hydrolysis signal generation.
  • multicolour fluorescent detection we favourably work on the channels with different ranges of a multichannel device, which channels, depending on the fluorescent labels used, are favourably the following: 522-537 nm / 555-567 nm / 602-615 nm / 632-647 nm / 665-775 nm / 700-710 nm.
  • This Fluorescence Shift extension towards the red spectrum is a safe solution from the aspect of quantitative evaluation, and by this the emission spectra of the individual real-time PCR dyes provide emission maximum in a wider range.
  • the emitted light intensity peak instead of the narrow wavelength range of ⁇ 10 nm, can be detected in a wider wavelength range between 20-35 nm.
  • the detection system can be repeated with appropriate precision on most real-time PCR platforms (Roche, ABI, BioRad, Corbett, Stratagene, etc.) available on the market, and it ensures a reaction with excellent fluorescence characteristics.
  • the multichannel fluorescent detection according to our procedure can be performed without fluorescence background optimisation requiring the use of ROX carboxy-X-rhodamine reference dye.
  • An example of the realisation of our procedure is the triplex tricolour real-time PCR determination of the Influenza A virus genotype version A/2009HlNlswl of swine origin, which is described in detail in our first implementation example.
  • Favourable realisations involve the 5' end iso-fluorescein-amino-methyl and 3' end iso-tetramethyl-NFQ, the 5' end iso- carboxyl-dichloro-dimethoxyfluorescein and 3' end NFQ, and the 5' end iso-carboxyl- dichloro-rhodamin and 3' end NFQ labelling of the probes.
  • Another example of a further realisation of our procedure is the determination of genotype version la/lb of the Hepatitis C virus, which is described in detail in the second implementation example.
  • 5' end fluorophore reporter and 3' end acceptor-quencher components are bound with covalent bonds.
  • Favourable realisations involve the 5' end iso-fluorescein- amino-methyl and 3' end iso-tetramethyl-NFQ, and the 5' end iso-carboxyl-dichloro- dimethoxyfluorescein and 3' end NFQ labelling of the probes.
  • the quantitative determination is based on subtype characteristic unique variable gene sequences occurring in one single copy in the viral genome examined.
  • An example of this is presented in our second implementation example and in figure 5 describing the analysis and quantitative determination of genotype la/ lb of Hepatitis C virus using duplex dual-colour real-time RT-PCR method.
  • the invention relates to a procedure for the rapid determination of Influenza A virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral R A in different samples taxonomically characteristic M2 matrix protein coding stable consensus m2 and 2009 swine zoonoses subtype characteristic HA/HI haemagglutinin lectin coding hl/sw, NP nucleoprotein coding np/sw unique variable nucleotide sequences are detected simultaneously using triplex tricolour real-time PCR technique.
  • the PCR probe element oligonucleotide primer pairs forward - reverse
  • the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation favourably suit the SEQ ID NO 1 - SEQ ID NO 2, SEQ ID NO 4 - SEQ ID NO 5, SEQ ID NO 7 - SEQ ID NO 8 and the SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9 sequences planned by us (see sequence listing).
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 (see sequence listing).
  • the invention also relates to a procedure for the rapid determination of Dengue virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, during the joint examination of serotypes DV1, DV2, DV3, DV4 of the virus, CP capsid polyprotein coding taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously using duplex dual-colour (four genotypes in 2 x dual-colour PCR reaction) real-time PCR technique.
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 25, favourably they suit sequences SEQ ID NO 14 - SEQ ID NO 15, SEQ ID NO 18 - SEQ ID NO 19, SEQ ID NO 22 - SEQ ID NO 23, SEQ ID NO 26 - SEQ ID NO 27 and sequences SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 24, SEQ ID NO 28 (see sequence listing).
  • the invention also relates to a procedure for the rapid determination of Hepatitis A virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, HsAg surface immunodeterminant coding taxonomically characteristic stable consensus VP-1 and subtype characteristic unique variable hsag-HA V nucleotide sequences are detected simultaneously using duplex dual-colour real-time PCR technique.
  • the detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention.
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 29, SEQ ID NO 33, favourably they suit sequences SEQ ID NO 30 - SEQ ID NO 31, SEQ ID NO 34 - SEQ ID NO 35 and SEQ ID NO 32, SEQ ID NO 36 (see sequence listing).
  • the invention also relates to a procedure for the rapid determination of Hepatitis B virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral DNA in different samples, for differentiating the eight most significant internationally recognised genotypes A-H of the virus, the current combination of the taxonomically characteristic core region and large S protein coding c stable consensus and subtype characteristic HB surface antigen coding s unique variable nucleotide sequences is detected simultaneously using duplex dual-colour real-time PCR technique.
  • the detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention.
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 37, SEQ ID NO 41, favourably they suit sequences SEQ ID NO 38 - SEQ ID NO 39, SEQ ID NO 42 - SEQ ID NO 43 and SEQ ID NO 40, SEQ ID NO 44 (see sequence listing).
  • the invention also relates to a procedure for the rapid determination of Hepatitis C virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, during the determination of the six different internationally recognised genotypes la/lb, 2a, 3a, 4a, 5a, 6a of the virus, taxonomically characteristic 5' end UTR untranscribing non-coding stable consensus sequences and subtype characteristic NSP non-structural protein coding sequences are detected simultaneously using duplex dual-colour real-time PCR technique.
  • the PCR probe element oligonucleotide primer pairs forward - reverse
  • the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation favourably suit sequences SEQ ID NO 45 - SEQ ID NO 46, SEQ ID NO 48 - SEQ ID NO 49 and sequences SEQ ID NO 47, SEQ ID NO 50 planned by us (see sequence listing).
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 51, SEQ ID NO 52 (see sequence listing).
  • the invention also relates to a procedure for the rapid determination of Human immunodeficiency virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, for the differentiation of the two most significant internationally recognised genotypes HIV-I, HIV-II of the virus, taxonomically characteristic nef envelope protein coding stable consensus and subtype characteristic gag nucleocapsid coding unique variable nucleotide sequences are detected simultaneously using duplex dual-colour real-time PCR technique.
  • the detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention.
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 53, SEQ ID NO 57, favourably they suit sequences SEQ ID NO 54 - SEQ ID NO 55, SEQ ID NO 58 - SEQ ID NO 59 and sequences SEQ ID NO 56, SEQ ID NO 60 (see sequence listing).
  • the invention also relates to a procedure for the rapid determination of Human papillomavirus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral DNA in different samples, taxonomically characteristic E6 early protein coding stable consensus e6 and subtype characteristic Ll-L low-risk factor protein coding or Ll-H high-risk factor protein coding unique variable proto-oncogene nucleotide sequences are detected simultaneously using duplex dual-colour real-time PCR technique.
  • sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention.
  • the templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 61, SEQ ID NO 65, SEQ ID NO 69, favourably they suit sequences SEQ ID NO 62 - SEQ ID NO 63, SEQ ID NO 66 - SEQ ID NO 67, SEQ ID NO 70 - SEQ ID NO 71 and sequences SEQ ID NO 64, SEQ ID NO 68, SEQ ID NO 72 (see sequence listing).
  • the invention also relates to a KIT enabling the practical realisation of our ' procedure aimed at the rapid determination of viruses using nucleic acid-based molecular diagnostics, a possible version of which is shown in figure 6.
  • Figure 1 Mechanism of fluorescent signal generation determined on the basis of the distance in space between PCR probe element fluorescent-labelled oligonucleotide probe 5' end R reporter and 3' end Q acceptor-quencher sections (complementary strand displacement hydrolysis). Due to the additional exonuclease activity of the thermostable DNA polymerase synthesising the new chain, the fluorescent-labelled oligonucleotide probe hybridised to the complementary template sequences is cleaved off the template hydrolytically, the 5' end R reporter and the 3' end Q acceptor-quencher labels move away from each other in space, and so the fluorescent signal is freed and can be detected by instruments.
  • Figure 2 An example of the variable surroundings around stable sequences.
  • variable nucleotide cassettes can be observed around the stable sequences.
  • Figure 3 The schematic drawing of the flexible oligonucleotide probe sensitive for spatial conformation according to the invention (see the description).
  • Top left insert the process curve of the duplex dual-colour PCR reactions according to our procedure.
  • Top right insert calibration curve produced with the help of instrument software for quantitative evaluation.
  • Table 1 Influenza A virus A/2009HlNlswl genotype determination, triplex tricolour single-step real-time RT-PCR reaction, 5x MasterMix components (see implementation example 1).
  • Table 2 Influenza A virus A/2009HlNlswl genotype determination, triplex tricolour single-step real-time RT-PCR reaction components for a final volume of 20 ⁇ (see implementation example 1).
  • Table 5 Hepatitis C virus HCV 1 a/lb genotype determination, duplex dual-colour single- step real-time RT-PCR reaction components for a final volume of 20 ⁇ (see implementation example 2).
  • Table 6 Hepatitis C virus HCV la/ lb genotype determination, duplex dual-colour single- step real-time RT-PCR reaction measuring parameters (see implementation example 2).
  • Table 7 Measuring parameters of the procedure according to the invention.
  • the reactions described in the examples are reactions optimised for capillary real-time PCR instrument (Roche Light Cycler® 2.0), which can also take place on other platforms using the possibility of fluorescence shift (see the description).
  • Body excretions e.g. nasal or throat excretion
  • blood serum are the most suitable sample sources. Sampling takes place in compliance with the public health and epidemiological prescriptions.
  • Samples to be processed within 12 hours are stored in the dark, at a temperature between +2 °C and +8 °C. Samples to be processed later than this are to be stored at a temperature between -80 °C and -20 °C.
  • Nucleic acid isolation is performed using DNA or RNA isolating kits available in commercial distribution.
  • the Influenza A virus and Hepatitis C virus nucleic acid extraction is realised with RNA isolating kits (for example Roche High Pure Viral RNA kit, QIAamp Viral RNA Mini Kit).
  • the concentration and purity of the isolated RNA is examined before further applications (for example laboratory UV-visible spectrophotometry).
  • the isolated RNA must be stored at a temperature between -80 °C and -20 °C.
  • the carrier nucleic acid for efficient isolation and the following amplification it is favourable to use the carrier nucleic acid, in a final concentration of 1 ⁇ g/ ⁇ l.
  • Example 1 Influenza A virus A/2009HINlswl genotype detection using triplex tricolour single-step real-time RT-PCR technique
  • SEQ ID NO 1 - SEQ ID NO 2 From the planned, lyophilised SEQ ID NO 1 - SEQ ID NO 2, SEQ ID NO 4 - SEQ ID NO 5, SEQ ID NO 7 - SEQ ID NO 8 primer pairs and from the SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9 probes 100 pmol/ ⁇ stock solution is made by adding PCR grade water of a quantity determined on the accompanying synthesis report.
  • the probe is labelled with light-sensitive fluorescence dye.
  • the probe is labelled with light-sensitive fluorescence dye.
  • Table 2 shows how the triplex tricolour real-time PCR reaction of the Influenza A virus is set up, for a volume of 20 ⁇ per reaction. Following the order determined in table 2 measure together PCR grade water, 5x MasterMix buffer solution prepared according to table 1 , without enzymes, a mixture of RT recombinant MMLV Moloney Murine Leukemia Virus reverse transcriptase and recombinant TAQ Thermus aquaticus thermostable DNA polymerase enzymes (enzyme blend), and finally template RNA isolated from the sample. Make sure that the final concentration of the template RNA does not exceed the critical 1-25 nM value.
  • Example 2 Hepatitis C virus la/ lb genotype determination using duplex dual- single-step real-time RT-PCR technique
  • the probe is labelled with light-sensitive fluorescent dye.
  • light-sensitive fluorescent dye During the work processes, in order to make sure that the fluorescence shift takes place, special attention must be paid to that it is possibly exposed to light of a low intensity for a time as short as possible.
  • Table 5 shows how the duplex dual-colour real-time PCR reaction of Hepatitis C virus is set up, for a volume of 20 ⁇ per reaction. Following the order determined in table 5 measure together PCR grade water, 5x MasterMix buffer solution prepared according to table 4, without enzymes, a mixture of RT recombinant MMLV Moloney Murine Leukemia Virus reverse transcriptase and recombinant TAQ Thermus aquaticus thermostable DNA polymerase enzymes (enzyme blend), and finally template RNA isolated from the sample. Make sure that the final concentration of the template RNA does not exceed the critical 1-25 nM value.

Abstract

Procédé de détermination rapide de virus à l'aide de diagnostics moléculaires basés sur des acides nucléiques, pour la détermination qualitative et quantitative de virus à ADN ou de virus à ARN dans des échantillons par la technique de la PCR multiplex en temps réel, au cours de laquelle dans des environnements génétiques variables, des séquences consensus stables toxinomiquement caractéristiques et des séquences nucléotidiques variables uniques sont détectées simultanément. Pour ce faire, des sondes oligonucléotidiques simple brin contenant des motifs palindromiques sont créées. Dans ces motifs palindromiques, des bases semi-dégénérées sont insérées en fonction des régions variables du virus en cours de détection, de préférence, des bases avantageuses en fonction des séquences variables exprimées en nombre dans les protocoles professionnels internationaux, lesdites bases semi-dégénérées permettant de former de multiples boucles internes pour garantir la flexibilité structurale des sondes. L'étroite proximité de type ponts en butée desdites multiples boucles internes comprend des séquences nucléotidiques avec lesquelles les sondes sont liées sur des séquences nucléotidiques stables de la matrice à détecter, accessibles dans les bases de données génétiques internationales, par un phénomène de forte hybridation thermodynamique. Sous l'effet de cette forte hybridation thermodynamique, lesdites multiples boucles internes s'ouvrent, par enjambement souple des régions variables autour des séquences stables de la matrice, les sondes se lient aux séquences variables, et sur la matrice à détecter, une séquence d'une longueur d'au moins 25-30 nucléotides liés d'une manière thermodynamiquement stable et sélective est détectée. Cette invention concerne également une trousse pour la mise en œuvre pratique dudit procédé.
PCT/HU2011/000045 2011-05-11 2011-05-11 Procédé de détermination rapide de virus à l'aide de diagnostics moléculaires basés sur des acides nucléiques, et trousse pour sa mise en œuvre WO2012153153A1 (fr)

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PCT/HU2011/000045 WO2012153153A1 (fr) 2011-05-11 2011-05-11 Procédé de détermination rapide de virus à l'aide de diagnostics moléculaires basés sur des acides nucléiques, et trousse pour sa mise en œuvre

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US20150225802A1 (en) * 2012-10-04 2015-08-13 Board Of Trustees Of The Leland Standford Junior University Methods and Reagents for Detection, Quantitation, and Serotyping of Dengue Viruses
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CN107083449A (zh) * 2017-05-08 2017-08-22 英科新创（苏州）生物科技有限公司 一种丙型肝炎病毒基因分型定量检测试剂盒
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CN113736867A (zh) * 2021-09-03 2021-12-03 广西国际旅行卫生保健中心(南宁海关口岸门诊部) 一种同时用于伊蚊分类监测和登革病毒检测的试剂盒

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