WO2017081560A1 - Kit et procédé in vitro pour le diagnostic précoce de la leucoencéphalopathie multifocale progressive - Google Patents

Kit et procédé in vitro pour le diagnostic précoce de la leucoencéphalopathie multifocale progressive Download PDF

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Publication number
WO2017081560A1
WO2017081560A1 PCT/IB2016/055715 IB2016055715W WO2017081560A1 WO 2017081560 A1 WO2017081560 A1 WO 2017081560A1 IB 2016055715 W IB2016055715 W IB 2016055715W WO 2017081560 A1 WO2017081560 A1 WO 2017081560A1
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WIPO (PCT)
Prior art keywords
seq
primer
homology
sequence
pcr
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PCT/IB2016/055715
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English (en)
Inventor
Luca ESPOSITO
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Euroviron S.R.L.
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Publication of WO2017081560A1 publication Critical patent/WO2017081560A1/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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention concerns a PCR kit comprising four primers and an in vitro method for the early diagnosis of progressive multifocal leukoencephalopathy (PML) .
  • PML progressive multifocal leukoencephalopathy
  • the John Cunningham virus is an ubiquitous human polyomavirus which infects over 80% of the human population during infancy. Generally the JCV persists in a latent state in different cell types, in which it has a stable archetypal control region .
  • the immunological alterations resulting from immunosuppressive pathologies or from the administration of monoclonal antibodies create an environment that allows JCV reactivation and infection associated with the development of progressive multifocal leukoencephalopathy (PML) , a demyelinating pathology of the central nervous system with fatal outcome.
  • PML progressive multifocal leukoencephalopathy
  • the best strategy for limiting the development of PML comprises early identification of the pathology on the basis of first indications of JCV replication or reactivation.
  • JCV is characterized by a genome having circular DNA with double strand of approximately 5130 base pairs (bp) .
  • the viral DNA is divided into three main regions: early region, non-coding region and late region.
  • the early region consists of 2400 bp and codes for 5 proteins: large (T) tumor antigen, small (t) tumor antigen, three splicing variants T135, T136 and T165.
  • NCCR non-coding control region
  • HVRR hypervariable regulatory region
  • the NCCR is the most variable region and its variations are associated with a viral tropism, a transcription and a replication which are different in patients with PML.
  • Different variants are currently known which are divided into archetype form and prototype sequences.
  • the archetype form is not associated with the development of PML, but appears to be at the origin of the prototype variants resulting from numerous rearrangements of the NCCR region, for example deletions and/or duplications of the original archetypal sequence.
  • the late region consists of 2300 bp, and codes for viral proteins of the capsid, the VP1 (the main protein of the capsid which allows viral entry into the human host cells and contains the epitopes for antibody induction and recognition) , the VP2 and VP3 (the minor proteins of the capsid necessary for JCV propagation) , and the agnoprotein (the smallest protein of JCV, which is involved in the late phase of the infection) .
  • the VP1 the main protein of the capsid which allows viral entry into the human host cells and contains the epitopes for antibody induction and recognition
  • the VP2 and VP3 the minor proteins of the capsid necessary for JCV propagation
  • the agnoprotein the smallest protein of JCV, which is involved in the late phase of the infection
  • US 2014/0255915 describes a diagnostic assay based on amplification by means of real-time polymerase chain reaction (PCR) of a conserved sequence of JCV. It has been verified, however, that this sequence is homologous not only among different strains of JCV, but also among different viral species (for example virus BK and SV40) .
  • the assay described in US 2014/0255915 therefore does not allow a reliable discrimination of the JCV from other viral species and of JCV strains that cause and do not cause PML .
  • An object of the present invention is therefore to provide a PCR kit which solves the above-mentioned problems in a simple and efficient manner.
  • a further object of the present invention is to provide a method as defined in claim 5.
  • the kit according to the present invention comprises four primers.
  • the first primer comprises a sequence having at least 90% homology with SEQ ID NO:l, preferably at least 95% homology with SEQ ID NO : 1 , more preferably at least 99% homology with SEQ ID NO:l, even more preferably it comprises SEQ ID NO:l.
  • the second primer comprises a sequence having at least 90% homology with SEQ ID NO : 2 , preferably having at least 95% homology with SEQ ID NO: 2, more preferably at least 99% homology with SEQ ID NO: 2, even more preferably it comprises SEQ ID NO: 2.
  • the third primer comprises a sequence having at least 90% homology with SEQ ID NO: 3, preferably having at least 95% homology with SEQ ID NO: 3, more preferably having at least 99% homology with SEQ ID NO: 3, even more preferably it comprises SEQ ID NO: 3.
  • the fourth primer comprises a sequence having at least 90% homology with SEQ ID NO : 4 , preferably at least 95% homology with SEQ ID NO: 4, more preferably at least 99% homology with SEQ ID NO: 4, even more preferably it comprises SEQ ID NO: 4.
  • the first primer is SEQ ID NO:l
  • the second primer is SEQ ID NO: 2
  • the third primer is SEQ ID NO : 3
  • the fourth primer is SEQ ID NO: 4.
  • the kit comprises a first probe and a second probe and the PCR is a real-time PCR.
  • the first probe comprises a sequence having at least 90% homology with SEQ ID NO: 5, preferably at least 95% homology with SEQ ID NO: 5, more preferably at least 99% homology with SEQ ID NO: 5, even more preferably it comprises SEQ ID NO: 5.
  • the second probe comprises a sequence having at least 90% homology with SEQ ID NO: 6, preferably at least 95% homology with SEQ ID NO: 6, more preferably at least 99% homology with SEQ ID NO: 6, even more preferably it comprises SEQ ID NO: 6.
  • the first probe is SEQ ID NO: 5 and the second probe is SEQ ID NO: 6.
  • the first and the second probe are marked so that they can be detected by means of real-time PCR.
  • Real-time PCR is now a well consolidated technique and therefore will not be described in detail here. See, for example, McKay et al . , Real-time PCR in virology, Nucl . Acids. Res. 2002, 20:1292.
  • the primers with SEQ ID NO:l and SEQ ID NO: 2 form a set of primers which selectively amplifies a sequence of the NCCR of the archetypal form of JCV, while the primers with SEQ ID NO: 3 and SEQ ID NO: 4 form a set of primers which selectively amplify a sequence of the gene for the protein VP1 which is homologous in all the JCV variants.
  • the primers can be designed using the Primer 3 software ( Simgene . com) .
  • the amplification by means of the first and second primer gives rise to an amplicon which identifies the presence of archetypal JCV, i.e. the non ⁇ pathogenic strain.
  • the amplification by means of the third and fourth primer gives rise to an amplicon which discriminates the presence of JCV and quantifies it. It should be noted that there are over ten pathogenic strains of JCV, i.e. those that cause PML, and they are difficult to identify because they are extremely variable.
  • the in vitro method for early diagnosis of progressive multifocal leukoencephalopathy (PML) comprises a first step of extracting the DNA from a biological sample and a second step of PCR on the extracted DNA.
  • the PCR uses the four primers of the kit described above.
  • the PCR is a real-time PCR and the first probe and the second probe described above are used.
  • the PCR is preferably performed using a first heating step ranging from 94°C to 96°C (more preferably at 95°C) for 8-12 minutes (more preferably 10 minutes), a second heating step ranging from 94°C to 96°C (more preferably at 95°C) for 14 to 16 seconds (more preferably 15 seconds), a third heating step ranging from 59°C to 61°C (more preferably at 60°C) ranging from 0.5 minutes to 1.5 minutes (more preferably for one minute), repeating the second step and the third step 35 to 43 times (more preferably 39 times) .
  • the biological sample from which the DNA is extracted is preferably cerebrospinal fluid or urine of a human individual. Examples
  • DNA of 8 multiple sclerosis (MS) patients and of 8 patients positive to PML was extracted from samples of urine and cerebrospinal fluid respectively.
  • the Nucleospin RNA virus kit (Macherey Nagels, Duren, Germany) was used, according to the manufacturer's instructions.
  • JCV DNA The presence and quantity of JCV DNA in each sample was determined by means of real-time PCR using the Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems) .
  • the primers and the probes were designed by means of the Primer 3 software and synthesized by Primm srl (for the primers) and Applied Biosystems (for the probes) .
  • the real-time PCR was performed using the Taqman Universal PCR Master Mix (Applied Biosystems) and each reaction was prepared as follows.
  • a quantitative multiplex PCR was performed which amplifies and identifies both a sequence of the NCCR of the archetypal form of JCV, and a sequence of the gene for the protein VP1.
  • the probe of SEQ ID NO: 5 is marked at the 5' end with NED and at the 3' end with MinorGrooveBinder .
  • the probe of SEQ ID NO: 6 is marked at the 5' end with VIC and at the 3' end with MinorGrooveBinder.
  • MS multiple sclerosis
  • the kit and the method according to the present invention therefore allow extremely accurate diagnosis of PML.

Abstract

La présente invention concerne un kit de PCR comprenant une première amorce comprenant une séquence ayant au moins 90 % d'homologie avec SEQ ID NO: 1, une deuxième amorce comprenant une séquence ayant au moins 90 % d'homologie avec SEQ ID NO: 2, une troisième amorce comprenant une séquence ayant au moins 90 % d'homologie avec SEQ ID NO : 3 et une quatrième amorce comprenant une séquence ayant au moins 90 % d'homologie avec SEQ ID NO : 4. La présente invention concerne en outre un procédé in vitro de diagnostic précoce de la leucoencéphalopathie multifocale progressive (LEMP) qui met en œuvre ces amorces.
PCT/IB2016/055715 2015-11-10 2016-09-23 Kit et procédé in vitro pour le diagnostic précoce de la leucoencéphalopathie multifocale progressive WO2017081560A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITUB2015A005426A ITUB20155426A1 (it) 2015-11-10 2015-11-10 Kit e metodo in vitro per la diagnosi precoce di leucoencefalopatia multifocale progressiva
IT102015000071052 2015-11-10

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WO2017081560A1 true WO2017081560A1 (fr) 2017-05-18

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013192100A1 (fr) * 2012-06-18 2013-12-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Procédés et compositions pour la détection du virus jc
WO2014170453A1 (fr) * 2013-04-18 2014-10-23 Janssen Diagnostics Bvba Analyse quasi-espèce de l'adn de virus jc présent dans l'urine de sujets sains

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013192100A1 (fr) * 2012-06-18 2013-12-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Procédés et compositions pour la détection du virus jc
WO2014170453A1 (fr) * 2013-04-18 2014-10-23 Janssen Diagnostics Bvba Analyse quasi-espèce de l'adn de virus jc présent dans l'urine de sujets sains

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CAROLINE F. RYSCHKEWITSCH ET AL: "Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants", JOURNAL OF CLINICAL VIROLOGY, vol. 57, no. 3, 1 July 2013 (2013-07-01), NL, pages 243 - 248, XP055305322, ISSN: 1386-6532, DOI: 10.1016/j.jcv.2013.03.009 *
ELENA ANZIVINO ET AL: "High Frequency of JCV DNA Detection in Prostate Cancer Tissues", CANCER GENOMICS & PROTEOMICS, vol. 12, no. 4, 1 July 2015 (2015-07-01), pages 189 - 200, XP055285696 *
FRISQUE R J ET AL: "HUMAN POLYOMAVIRUS JC VIRUS GENOME", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 51, no. 2, 1 August 1984 (1984-08-01), pages 458 - 469, XP008041561, ISSN: 0022-538X *
GUI PING HAN ET AL: "Genetic analysis of JC virus and BK virus from a patient with progressive multifocal leukoencephalopathy with hyper IgM syndrome", JOURNAL OF MEDICAL VIROLOGY, vol. 76, no. 3, 1 January 2005 (2005-01-01), US, pages 398 - 405, XP055285685, ISSN: 0146-6615, DOI: 10.1002/jmv.20377 *
REID C E ET AL: "Sequencing and analysis of JC virus DNA from natalizumab-treated PML patients", THE JOURNAL OF INFECTIOUS DISEASES UNITED STATES 15 DEC 2010, INFECTIOUS DISEASES SOCIETY OF AMERICA, US, vol. 204, no. 2, 15 July 2011 (2011-07-15), pages 237 - 244, XP002710479, ISSN: 1537-6613, DOI: 10.1093/INFDIS/JIR256 *
TOM VAN LOY ET AL: "Quasispecies Analysis of JC Virus DNA Present in Urine of Healthy Subjects", PLOS ONE, vol. 8, no. 8, 15 August 2013 (2013-08-15), pages e70950, XP055285703, DOI: 10.1371/journal.pone.0070950 *

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