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WO2011098775A1 - Source d'acide nucléique non infectieuse - Google Patents

Source d'acide nucléique non infectieuse Download PDF

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WO2011098775A1
WO2011098775A1 PCT/GB2011/000195 GB2011000195W WO2011098775A1 WO 2011098775 A1 WO2011098775 A1 WO 2011098775A1 GB 2011000195 W GB2011000195 W GB 2011000195W WO 2011098775 A1 WO2011098775 A1 WO 2011098775A1
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virus
nucleic acid
rna
sequence
modified
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George Peter Lomonossoff
Nicholas Montague
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Plant Bioscience Ltd
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Plant Bioscience Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/32043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to methods and materials for use in preparing nucleic acid such as RNA, for example for use as standards or controls in nucleic acid analysis.
  • RNA transcripts that do not require a lysis step to release nucleic acid template only provide limited validation of the RNA extraction process of an assay.
  • the development of control RNA molecules which are encapsidated in particles is one strategy that can be deployed to replicate more closely the conditions encountered by the RNA template of wild-type virus within clinical samples.
  • WO2007/104120 describes the use of a calibration virus to act as a control for HIV viral load tests in real time PCR.
  • the invention uses modified HIV viruses as a calibration for assessing clinical HIV.
  • US2007/0077554 concerns the use of homologous viral controls for RT PCR i.e. controls derived from the viruses which they are intended to be used with.
  • W095/34684 concerns tagged viral standards, particularly infectious ones.
  • CPMV Cowpea Mosaic Virus
  • a cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of CPMV. After co-inoculation with a plasmid that expressed CPMV RNA-1 , recombinant virus particles were isolated from cowpea (Vigna unguiculata) plants.
  • RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results.
  • the recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target.
  • This system provided an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and the purification method is straightforward. Though highly satisfactory as a method for producing controls for PCR analysis, the method reported by King et al.
  • RNA1 -containing particles Furthermore the presence of only a very few numbers of RNA1 -containing particles enabled infectivity to be regained.
  • US 5919625 (Ambion) relates to processes for creating a recombinant nucleic acid standard which is resistant to ribonuclease digestion and is non-infectious.
  • a single strand of recombinant nucleic acid is encapsidated by bacteriophage proteins.
  • the recombinant nucleic acid is a hybrid sequence encoding bacteriophage proteins and a specific non-bacteriophage sequence.
  • a non-bacteriophage RNA sequence can be used as an RNA standard to help quantify the number of RNA molecules in an unknown sample.
  • the recombinant RNA in its packaged form is highly resistant to ribonucleases, insuring that the RNA standard is not compromised by inadvertent ribonuclease contamination. Disclosure of the invention
  • the present inventors appreciated a need for efficiently producing viral particles which are not able to spread infection and that can be used as standards for the analysis of nucleic acid, for example for PCR.
  • This invention provides a solution to the issues discussed above in relation to King et al 2007, by providing a method for the production of preparations of viral particles, for example CPMV particles, which are compromised in their ability spread infection for use as diagnostic controls. A refined method for the insertion of diagnostic cassettes is also provided.
  • the invention provides a method for efficiently producing viral particles which are not able to spread infection by providing CPMV and other viruses which have a deleted or otherwise non-functional movement protein.
  • WO2007/135480 Unlike earlier publications concerning modified CPMV, such as WO2007/135480, the formation of viral particles is a key part of the present invention.
  • the formation of virus particles is prevented by deletion of the coat protein encoding region of the virus.
  • methods for making a reference standard for nucleic acid-based assays which comprises preparing a movement protein deleted virus, and optionally inserting into said virus a target sequence of interest.
  • the invention thus provides a process for preparing a target or reference nucleic acid for use in a nucleic-acid based assay, which process comprises:
  • the invention provides a process for preparing a target or reference nucleic acid for use in a nucleic-acid based assay, which process comprises:
  • the invention provides a process for preparing a target or reference nucleic acid for use in a nucleic-acid based assay, which process comprises:
  • the invention may be performed using both DNA or RNA genome viruses.
  • the process comprises use of DNA encoding the viral genome.
  • Such method may include (i) providing DNA encoding a viral RNA genome which has been modified such that cell to cell movement of the encoded virus has been compromised;
  • compromised virus comprising said reference RNA is produced.
  • step (iii) may comprise:
  • RNA2 and RNA1 are produced in the host cells in viral-encapsulated form e.g. by culturing the cells under conditions allowing for transcription of the recombinant RNA segment and encapsidation of the recombinant RNA segment in viral coat protein.
  • the process comprises: (i) providing an RNA genome virus which has been modified such that cell to cell movement of the virus has been compromised and into which an RNA-reference sequence has been introduced,
  • the reference nucleic acid optionally still encapsulated, so produced may be used in the nucleic acid based assay.
  • the process may further comprise:
  • nucleic acid based assay using said reference nucleic acid (e.g. RNA, optionally still encapsulated, or isolated).
  • reference nucleic acid e.g. RNA, optionally still encapsulated, or isolated.
  • the assay is performed using the reference nucleic acid in encapsulated form, and the particles are mixed with the test sample and assayed together in a One pot' procedure. Initially, the encapsidation of the RNA protects it from any nucleases in the sample. Thereafter, those skilled in the art are able to readily extract the RNA from the 'doped' sample (for example using commercially available reagents such as TRIzol® available from Invitrogen) and assess the presence of target RNA and the (positive) control sequence.
  • the assay will utilise primers which amplify both the target sequence from the sample, and also the reference nucleic acid (i.e. the reference nucleic acid includes the same primer binding sites as the target).
  • any amplification product of the target from the sample is distinguishable from that resulting from the reference sequence e.g. in terms of sequence or length.
  • Reference nucleic acid, or modified viruses, obtained by the processes described herein form further aspects of the invention, as do uses of the same in nucleic acid assays.
  • use of a virus which has been modified such that cell to cell movement has been compromised for producing a reference standard for nucleic acid-based assays.
  • a kit for performing a nucleic-acid based assay comprises DNA encoding a viral genome which has been modified such that cell to cell movement of the encoded virus has been compromised.
  • the DNA includes a reference (or reference-encoding) DNA sequence.
  • kit for performing a nucleic-acid based assay comprises a virus which has been modified such that cell to cell movement of the virus has been compromised and optionally into which an RNA-reference sequence has been introduced.
  • the reference sequence is conveniently introduced into the viral genome (i.e. is heterologous to it).
  • the reference sequence e.g. RNA-reference-encoding DNA sequence
  • the reference sequence is in a non-translated part of the viral genome.
  • the modification is to the 'movement protein' of the virus.
  • the virus is a Comovirus, such as CPMV or CCMV.
  • the nucleic-acid based assay may for example be a quantitative, semi-quantitative or qualitative RNA-based assay e.g. RT-PCR. Accordingly, it is one object of this invention to provide compositions for use as nucleic acid assay standards, which compositions provide good internal standards and which are not able to spread infection. As explained below, the reference nucleic acid is
  • Figure 1 shows use of wild-type CPMV as a positive control in a PCR reaction at a dilution in excess of 16 million fold.
  • Figure 2 shows use of movement protein deleted CPMV with and without a test target sequence (AIV) as a PCR positive control at a dilution of one million fold.
  • AIV test target sequence
  • Figure 3 shows the cloning strategy used in the Examples below. As shown in the Figure, firstly - plasmid CVW Apal is cut with BamHI and EcoRI to give a BamHI and
  • Plasmid pN81 S2NT was cut with Ndel and AccI, polished with Klenow fragment and then re-ligated. This had the effect of cutting out a 456 Bp fragment but maintaining everything in-frame.
  • This plasmid is referred to as pN81 S2NTMPdel and contains the deleted movement protein.
  • a Pad / BamHI fragment encoding the deleted movement protein is then excised from pN81S2NTMPdel and ligated into plasmid pBIN-mim to give pBIN-mim-MPdel.
  • Figure 4 illustrates an embodiment of the invention including an insert corresponding to the FMDV genome.
  • FIG. 5 illustrates the results obtained using the embodiment in Figure 4.
  • Figure 6 illustrates how control and the target sequences can be distinguished through the use of differential probes linked to differing fluorescent reporters.
  • Cowpea Mosaic Virus is a non-enveloped RNA plant virus classified in the Family Comoviridae within the proposed new order Picornavirales. CPMV shares some similarities with the genome organisation and capsid structure of picornaviruses such as FMDV and SVDV. However, CPMV has a bipartite genome: each of the single-stranded positive sense genomic segments are packaged separately into mature icosahedral viral capsids and each encodes a polyprotein. RNA-1 encodes the proteins involved in protein processing and RNA replication.
  • RNA-1 self-processes in cis through the action of the 24K proteinase domain to give the 32K proteinase co-factor, the 58K helicase, the VPg, the 24K proteinase and the 87K RNA-dependent RNA-polymerase.
  • RNA-2 is translated to give a pair of polyproteins,(the 105K and 95K proteins) as a result of initiation at two different AUG codons at positions 161 and 512. These polyproteins are processed by the RNA-1 -encoded 24K proteinase in trans at 2 sites to give the 58K/48K pair of proteins (which differ only at their N-terminus) and the mature L and S coat proteins.
  • CPMV in order to cause an infection of plants, CPMV must not only be able to replicate in individual cells but must also be able to spread to neighbouring cells. Without the ability to spread, an infection remains "subliminal", being restricted to only those cells which initially received the virus.
  • the ability of CPMV to spread is governed by the 48K protein produced by RNA-2. This protein produces tubules which penetrate the plasmodesmata of infected cells and through which virus particles traffic (van Lent et al., 1990).
  • RNA-2 of CPMV was excised. After filling-in the 5' overhangs using E.coli Poll (Klenow fragment), the DNA was re-ligated resulting in a construct in which 456 nucleotides encoding the central region of the 48K protein, encompassing regions deemed essential by Bertens et al. (2000), were deleted.
  • capsids were recoverable, they were not able to establish infections in plants and serve as excellent controls for PCR reactions.
  • compositions of matter are available for use in diagnostic methods.
  • CPMV Comoviridae
  • CCMV Comoviridae
  • the encapsidated reference sequences or "internal controls" of the present invention may be used in similar manner analogously to those in the art. See for example Hoffman et al 2009 "A review of RT-PCR technologies used in veterinary virology and disease control: Sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health” Veterinary Microbiology 139
  • viral particles comprising (protected) reference nucleic molecules can be used as standards in any desired nucleic acid-based assay.
  • examples are well known to those skilled in the art and include real time and ⁇ or quantitative RT-PCR, or isothermic assays such as LAMP.
  • the present invention is particularly useful for viral quantification.
  • nucleic acid based assays in the process of being developed and/or marketed (Roche Diagnostic Systems, AMPLICOR.TM. HIV Monitor and AMPLICOR.TM. HCV Monitor tests; Organon Teknika, NASBA HIV kit; GENPROBE, Transcription Mediated
  • Amplification HIV kit Amplification HIV kit
  • Chiron Corp. branched DNA (bDNA) signal amplification assay for HIV and HCV.
  • bDNA branched DNA
  • the target or reference nucleic acid typically RNA
  • the reference nucleic acid will typically correspond to part of the genome of that e.g. virus, and will be compatible with (comprise binding sites for) primer or probe sequences used in the assay.
  • pathogens of interest include HIV-1 , HIV-2, HCV, HTLV-1 , HTLV- 2, hepatitis G, enteroviruses, or a blood-borne pathogens.
  • pathogens of interest include HIV-1 , HIV-2, HCV, HTLV-1 , HTLV- 2, hepatitis G, enteroviruses, or a blood-borne pathogens.
  • RNA associated with a condition or cell type e.g. a hyperproliferative condition
  • a typical assay for the presence of an RNA of interest in a sample may comprise:
  • the reference sequence may be used as a positive control or internal standard in detection or quantification of the RNA of interest.
  • Preferred embodiments comprise PCR analysis.
  • quantitative assays will comprise comparing an amount of tested RNA PCR product with an amount of reference RNA PCR product.
  • the tested nucleic acid is often an RNA.
  • PCR analysis will usually comprise: (1) employing a reverse transcription procedure; (2) amplifying PCR product; and (3) detecting PCR product.
  • the amplification step involves co-amplification of any tested RNA PCR product with standard reference RNA PCR product.
  • co-amplification can be achieved via the use of a single primer set adapted for amplification of both tested RNA PCR product and reference RNA PCR product.
  • amplification of the control sequences may require that an additional set of primer pairs are included in the reaction.
  • 'mimic' ICs here: disabled, chimeric virus particles
  • these mimic ICs can contain internal sequences not present in the target amplicon to yield an amplification product with a different length which can be differentiated from wild-type amplicon by a second, IC-specific probe.
  • a benefit of this type of control is that it directly monitors the performance of diagnostic primer sets ensuring that the correct PCR components have been added.
  • the amplification product of the target and control are distinguishable by having different internal sequences, which in turn can be detected using different reporter sequences having distinguishable fluorescence characteristics.
  • RNA reference sequences with assay kits are given in US 5919625 discussed supra.
  • RNA plant viruses include, but are not limited to, other members of the family Comoviridae.
  • the genera of the Comoviridae family include Comovirus, Nepovirus, Fabavirus,
  • Comoviruses include Cowpea mosaic virus (CPMV), Cowpea severe mosaic virus (CPS V), Squash mosaic virus (Sq V), Red clover mottle virus (RCMV), Bean pod mottle virus (BPMV). Also included is tobacco mosaic virus (TMV), potato virus X (PVX), cowpea chlorotic mottle virus (CCMV).
  • CPMV Cowpea mosaic virus
  • CPS V Cowpea severe mosaic virus
  • Sq V Squash mosaic virus
  • RCMV Red clover mottle virus
  • BPMV Bean pod mottle virus
  • TMV tobacco mosaic virus
  • PVX potato virus X
  • RNA-2 The sequences of the RNA-2 genome segments of these comoviruses and several specific strains are available from the NCBI database under the accession numbers listed in brackets: cowpea mosaic virus RNA-2 (NC_003550), cowpea severe mosaic virus RNA-2 (NC_003544), squash mosaic virus RNA-2 (NCJD03800), squash mosaic virus strain Kimble RNA-2 (AF059533), squash mosaic virus strain Arizona RNA-2
  • RNA-2 sequences available from bean rugose mosaic virus (BRMV; AF263548) and a tentative member of the genus Comovirus, turnip ringspot virus (EF191015).
  • DNA viruses Numerous sequences from the other genera in the family Comoviridae and other viruses (including DNA viruses) are also readily available to those skilled in the art. Thus DNA viruses may also be adapted by those skilled in the art in a similar manner to carry specific heterologous sequences and may also be rendered non-infectious by deletion or disruption of genes required for systemic movement and/or infectivity. Maize Streak virus is an example of a DNA virus which could be used in this way. Such DNA viruses could serve as controls for pathogens with DNA-based genomes.
  • CPMV RNA-2 is shorter than RNA-1 (3.5 kb in comparison to 5.9 kb respectively), there is space in this segment to insert additional foreign RNA sequences without affecting encapsidation. This property has been exploited previously to develop RNA-2 into a vector for the efficient expression of heterologous proteins (Gopinath et al., 2000; Mechtcheriakova et al., 2006; Monger et al., 2006) and translatable sequences in excess of 1 kb have been successfully incorporated (Monger et al., 2006). However its use for producing non-translated RNA standards in non-transmissible viral capsids has not previously been taught or suggested.
  • the diagnostic sequence is introduced following the 'S protein' such that it is not translated i.e. after a stop codon.
  • the diagnostic sequence may be introduced into a pre-existing heterologous sequence or region which facilitates such further cloning procedures.
  • the invention may conveniently be employed by manipulating the viral genome on encoding nucleic acid e.g. plasmids. These can then be introduced into a population of cells such that compromised virus comprising nucleic having said reference sequence is produced.
  • nucleic acid e.g. plasmids.
  • plasmids e.g. plasmids
  • compromised virus comprising nucleic having said reference sequence is produced.
  • conventional agrobacterium based system may be employed to introduce DNA into a plant e.g. via agroinoculation.
  • the genome is present on a plant binary vector e.g. pBin19 (see Frisch, D. A., L. W. Harris-Haller, et al. (1995). "Complete Sequence of the binary vector Bin 19.” Plant Molecular Biology 27: 405-409).
  • a sequence e.g. all or part of the viral genome
  • Non-translated sequence will be downstream of a stop codon. Sequences for transcription will generally be operably linked to promoter and terminator sequences.
  • an expression cassette comprising:
  • a viral genome (are part thereof - e.g. RNA2) which has been modified to reduce cell to cell movement (i.e. systemic movement and/or infectivity) and into which a non- translated heterologous sequence has been incorporated,
  • a terminator sequence As noted above the non-translated heterologous sequence may be located downstream of the stop codon present or introduced into the genome or genome segment.
  • the heterologous sequence may be a polylinker or multiple cloning site, i.e. a sequence which facilitates cloning of a gene encoding a protein of interest into the expression system. More preferably it is diagnostic reference sequence
  • the promoter used to drive the gene of interest will be a strong promoter.
  • Examples of strong promoters for use in plants include the cauliflower mosaic 35S protein.
  • CPMV CPMV
  • 456 nucleotides encoding the central region of the 48K protein was deleted to render it non-functional.
  • the effectiveness of the modification can be confirmed by assaying for systemic symptoms in the cells or organism into which the virus is introduced e.g. spread within the plant, when employing CPMV or the like.
  • the modification will preferably render the virus incapable of cell to cell movement and incapable of transmission to further plants.
  • Nucleic acid or a “nucleic acid molecule” as used herein refers, depending on context, to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
  • isolated nucleic acid Is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • a vector such as a plasmid or virus vector
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues).
  • An "isolated nucleic acid” (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • phrases "consisting essentially of when referring to a nucleic acid means a sequence having the properties of a given sequence.
  • the phrase includes the sequence per se and minor changes and ⁇ or extensions that would not affect the function of the sequence, or provide further
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage, viral or
  • Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • the constructs used will be wholly or partially synthetic. In particular they are
  • a vector according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
  • a binary vector system includes (a) border sequences which permit the transfer of a desired nucleotide sequence into a plant cell genome; (b) desired nucleotide sequence itself, which will generally comprise an expression cassette of (i) a plant active promoter, operably linked to (ii) the target sequence and ⁇ or enhancer as appropriate.
  • the desired nucleotide sequence is situated between the border sequences and is capable of being inserted into a plant genome under appropriate conditions.
  • the binary vector system will generally require other sequence (derived from A. tumefaciens) to effect the integration. Generally this may be achieved by use of so called "agro-infiltration” which uses Agrobacterium- mediated transient transformation.
  • T-DNA Agrobacterium tumefaciens to transfer a portion of its DNA
  • the T-DNA is defined by left and right border sequences which are around 21-23 nucleotides in length.
  • the infiltration may be achieved e.g. by syringe (in leaves) or vacuum (whole plants).
  • the border sequences will generally be included around the desired nucleotide sequence (the T-DNA) with the one or more vectors being introduced into the plant material by agro-infiltration.
  • “Expression cassette” refers to a situation in which a nucleic acid is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial or plant cell.
  • a “promoter” is a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double- stranded DNA).
  • “Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • This plasmid encodes a full length RNA2 sequence of CPMV.
  • An Apal site was introduced after the stop codon of the S (small coat protein) protein in order to facilitate insertion of the reference sequence. This was done by cutting with Pstl and Stul and inserting the oligo :-
  • This plasmid is referred to as pCVW Apal
  • plasmid pN81 S2 NT (see Liu et al., 2002, herein incorporated by reference) This plasmid also encodes a full length RNA2 sequence of CPMV.
  • This plasmid is referred to as pN81 mp del
  • Plasmid pBin-NS1 (ds red) also includes a full length RNA2 sequence of CPMV.
  • This oligo also included an Avrll site to facilitate further cloning procedures between Apal and Avrll.
  • This plasmid is pBIN-mimAIV-Avrll
  • CTGCAAA CATCTCCTAGGTGGCC
  • CCGGGACGTTT GTAGAGGATCCA pN81 mp del was then cut with Pad and BamHI to release the fragment with the reduced movement protein. This fragment was then ligated into similarly cut pBin-mim and pBin- mim AIV Avrll.
  • the plasmid pBIN-mim AIV Avrll mp del produced may then be used as the vector, following restriction digestion with Apal and Avrll, accepting alternative heterologous sequence for encapsidation into the virus particle.
  • EXAMPLE 2 Particles from a construct with a partially-deleted Movement protein:
  • a sequence containing the diagnostic target from avian Influenza Virus (supplied by the Veterinary Laboratories Agency) has been inserted into the movement protein-deleted version of CPMV.
  • the insert contains the sequence of the PCR primers used for amplification of the M-gene of AIV.
  • Sap samples from both the inoculated primary leaves and from the trifoliate leaves of inoculated plants were passaged to new cowpea plants. No symptoms were observed on either primary or trifoliate leaves of plants inoculated with the sap from plants originally inoculated with the MP-deleted constructs. Inoculation with sap from plants inoculated with wild-type CPMV produced clear infection symptoms, including distinct disease foci visible on the primary leaves and strong systemic symptoms. These observations were again confirmed by PCR testing for CPMV. These results show that the movement protein-deleted virus is non-transmissible.
  • the above tests were repeated using the host Nicotiana benthemiana and in every case the MP-deleted virus was again shown to be both incapable of cell to cell movement i.e. no systemic symptoms were present, and transmission to further plants was not possible.
  • the yield of virus per kilogramme wet weight of harvested leaf material is given here in grams of purified virus /kilogramme of leaf tissue weighed at harvest. After purification, all virus particles were sequenced to confirm the genetic integrity of the RNA within virus particles. No mutations were observed in any of the constructs. Wild type virus, with an intact movement protein but with no foreign insert may be expected to yield 1g particles/kg leaves. CPMV with the movement protein intact carrying the AlV sequence has been shown to give a yield of 0.2g/kg. This has been shown to act as an effective positive control when used at a dilution in excess of 16 million-fold. See Figure 1.
  • CPMV with the MP deleted both with and without the AlV sequence insert gave a yield of approx 0.0025g/kg. This is approximately a 1000-fold reduction in yield compared to wt virus containing the same insert. Due to the sensitivity of the assay for which these particles are being used as a positive control this is not a concern.
  • the MP-deleted AlV particles were shown to give similar results to the RNA positive control, this control being an unencapsulated RNA transcript containing the M-gene diagnostic region from AlV,, at a dilution of one million-fold. See Figure 2.
  • RNA plant viruses include, but are not limited to, other members of the family Comovi dae tobacco mosaic virus (TMV), potato virus X (PVX), cowpea chlorotic mottle virus (CCMV).
  • TMV Comovi dae tobacco mosaic virus
  • PVX potato virus X
  • CCMV cowpea chlorotic mottle virus
  • DNA viruses could also be adapted in a similar manner to carry specific heterologous sequences and may also be rendered non-infectious by deletion or disruption of genes required for systemic movement and/or infectivity.
  • Maize Streak virus is an example of a DNA virus which could be used in this way.
  • DNA viruses could serve as controls for pathogens with DNA-based genomes.
  • the movement protein can be rendered non-functional either by the deletion of all or essential parts of the gene, making insertions within essential regions of the genes, mutating specific amino acids required for its correct function or creating a frameshift within the gene either by the insertion of deletion of nucleotides.
  • RNA transcripts are used or even simply plasmid DNA containing the relevant diagnostic target sequence. Neither of these standards are ideal as the initial stages of the diagnostic test are not required, that is the extraction of the genetic material form within a virus capsid, and, in the case of a DNA standard, the reverse transcription step. In addition, the highly unstable RNA molecules must be transported at -70°C.
  • the encapsidated mimics are ideal robust positive controls for dissemination as reference standards for inter-laboratory performance evaluation.
  • the mimics are biologically safe, and very stable.
  • the virus particles are stable in solution up to 60°C and 50% DMSO, and the RNA molecules within them are not degraded in the presence of biological material at ambient temperature or at 37°C for over a month. They are highly resistant to RNAse treatment.
  • the encapsulated RNA is readily released by standard lysis protocols (e.g. RNAzol) and amplifies efficiently in all cases. See King et al. (2007).
  • Figure 4 illustrates how a modified CPMV particle can be prepared including an insert corresponding to the FMDV genome.
  • the RNA in each can be released and assayed, with the results being distinguishable using different fluorescent reporters.
  • Figure 5 illustrates hypothetical results obtainable using positive and negative samples, in addition to a "false negative" result.
  • the 'mimic' has identical primer-binding sequences to the diagnostic target but has a different internal reporter-probe binding sequence. This enables the particles to be used simultaneously with the diagnostic target .
  • a sample of the appropriate mimic is added at the beginning of the diagnostic test and then follows in every way the path of the diagnostic target.
  • the forward and reverse primers will amplify both the diagnostic target and the control.
  • the sequence between the priming sites differs between the target and the control amplification of the control and the target can be distinguished through the use of differential probes linked to differing fluorescent reporters. This is illustrated in Figure 6.
  • the lower case characters represent the primer binding sites, and specific fluorescent probe site (middle).
  • V324 5'UTR of BVDV2 strain 890 (engineered for primer optimum binding) showing the binding of primers V324 and V326 plus the two Taq an probes BVDV 1/2 and BVDV2 (all bold).
  • Example N1 insert sequence TGATAGTGTCTGTTATTATGCCATTGTATTTCAATACAGCCACAGCCCCATTGTCTGGACCAGAAA TTCCAATTGTCAACCAACTGGTGCCATCATGGCAAGCACTTGCTGACCAAGCAACAGACTCAAACG ACTAGCTCATGTACGAGTCATGTGCTGGCGCTGATATGCGAAAACACGGAAGACTGACGACTACTG CTGATGACG 7)
  • Example Rabies insert sequence TGATAGTGTCTGTTATTATGCCATTGTATTTCAATACAGCCACAGCCCCATTGTCTGGACCAGAAA TTCCAATTGTCAACCAACTGGTGCCATCATGGCAAGCACTTGCTGACCAAGCAACAGACTCAAACG ACTAGCTCATGTACGAGTCATGTGCTGGCGCTGATATGCGAAAACACGGAAGACTGACGACTACTG CTGATGACG 7)
  • Example Rabies insert sequence Example Rabies insert sequence:

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Abstract

La présente invention a pour objet des procédés et des matériaux destinés à être utilisés dans la préparation d'acide nucléique, de préférence d'ARN, par exemple en tant que normes ou témoins dans une analyse d'acides nucléiques. Selon un aspect, l'invention concerne un procédé de préparation d'un acide nucléique cible ou de référence destiné à être utilisé dans une analyse basée sur les acides nucléiques, lequel procédé comprend les étapes consistant : (i) à fournir la totalité ou une partie d'un génome viral qui a été modifié de telle sorte que le déplacement de cellule à cellule du virus codé ait été compromis (par exemple par mutation d'une protéine de déplacement) et dans lequel la séquence de référence, de préférence non traductible, a été introduite ; (ii) à introduire ledit génome modifié dans une population de cellules de telle sorte que le virus fragilisé encapsidant l'acide nucléique ayant ladite séquence de référence soit produit. Un virus préféré est le virus mosaïque du dolique à œil noir.
PCT/GB2011/000195 2010-02-12 2011-02-14 Source d'acide nucléique non infectieuse Ceased WO2011098775A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015118183A1 (fr) * 2014-02-10 2015-08-13 Amptec Gmbh Arn enveloppé
CN113025610A (zh) * 2019-12-09 2021-06-25 广西壮族自治区兽医研究所 可视化二重lamp鉴别禽白血病病毒和鸡传染性贫血病毒的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034684A1 (fr) 1994-06-10 1995-12-21 Georgetown University Virus de recombinaison et methode pour le quantifier
US5919625A (en) 1996-07-03 1999-07-06 Ambion, Inc. Ribonuclease resistant viral RNA standards
US20070077554A1 (en) 2005-09-30 2007-04-05 Parshionikar Sandhya U Homologous viral internal controls for use in RT-PCR assays of enteric viruses
WO2007104120A2 (fr) 2006-03-16 2007-09-20 Fundação Oswaldo Cruz - FIOCRUZ Virus d'étalonnage artificiel destiné à contrôler des tests de charge virale du vih par pcr en temps réel
WO2007135480A1 (fr) 2006-05-22 2007-11-29 Plant Bioscience Limited Système bipartite, procédé et composition permettant l'expression constitutive et inductible de niveaux élevés de protéines étrangères dans des plantes
WO2009087391A1 (fr) * 2008-01-08 2009-07-16 Plant Bioscience Limited Systèmes d'expression de protéines

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034684A1 (fr) 1994-06-10 1995-12-21 Georgetown University Virus de recombinaison et methode pour le quantifier
US5919625A (en) 1996-07-03 1999-07-06 Ambion, Inc. Ribonuclease resistant viral RNA standards
US20070077554A1 (en) 2005-09-30 2007-04-05 Parshionikar Sandhya U Homologous viral internal controls for use in RT-PCR assays of enteric viruses
WO2007104120A2 (fr) 2006-03-16 2007-09-20 Fundação Oswaldo Cruz - FIOCRUZ Virus d'étalonnage artificiel destiné à contrôler des tests de charge virale du vih par pcr en temps réel
WO2007135480A1 (fr) 2006-05-22 2007-11-29 Plant Bioscience Limited Système bipartite, procédé et composition permettant l'expression constitutive et inductible de niveaux élevés de protéines étrangères dans des plantes
WO2009087391A1 (fr) * 2008-01-08 2009-07-16 Plant Bioscience Limited Systèmes d'expression de protéines

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
ANON.: "Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, OIE", 2006, article "Validation and quality control of polymerase chain reaction methods used for the diagnosis of infectious diseases"
BALLAGI-PORDANY, A.; BELAK, S: "The use of mimics as internal standards to avoid false negatives in diagnostic PCR", MOL. CELL. PROBES, vol. 10, 1996, pages 159 - 164
BELAK, S.; THOREN, P.: "Molecular diagnosis of animal diseases: some experiences over the past decade", EXPERT REV. MOL. DIAGN., vol. 1, 2001, pages 434 - 443
BELD, M; MINNAAR, R.; WEEL, J.; SOL, C.; DAMEN, M.; VAN DER AVOORT, H.; WERTHEIM-VAN DILLEN, P.; VAN BREDA, A.; BOOM, R.: "Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control", J. CLIN. MICROBIOL., vol. 42, 2004, pages 3059 - 3064, XP002661685, DOI: doi:10.1128/jcm.42.7.3059-3064.2004
BERTENS, P.; WELLINK, J.; GOLDBACH, R .; VAN KAMMEN, A.: "Mutational analysis of the cowpea mosaic virus movement protein", VIROLOGY, vol. 267, 2000, pages 199 - 208, XP004436104, DOI: doi:10.1006/viro.1999.0087
CALLAHAN, J.D.; BROWN, F.; OSORIO, F.A.; SUR, J.H.; KRAMER, E.; LONG, G.W.; LUBROTH, J.; ELLIS, S.J.; SHOULARS, K.S.; GAFFNEY, K.L: "Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus", J. AM. VET. MED. ASSOC., vol. 220, 2002, pages 1636 - 1642, XP008102191, DOI: doi:10.2460/javma.2002.220.1636
DAS, A.; SPACKMAN, E.; SENNE, D.; PEDERSEN, J; SUAREZ, D.L.: "Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents", J. CLIN. MICROBIOL., vol. 44, 2006, pages 3065 - 3073, XP002501623, DOI: doi:10.1128/jcm.00639-06
FRISCH, D. A.; L. W. HARRIS-HALLER ET AL.: "Complete Sequence of the binary vector Bin 19", PLANT MOLECULAR BIOLOGY, vol. 27, 1995, pages 405 - 409, XP000654452, DOI: doi:10.1007/BF00020193
GOPINATH, K.; WELLINK, J.; PORTA, C.; TAYLOR, K.M.; LOMONOSSOFF, G.P.; VAN KAMMEN, A.: "Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants", VIROLOGY, vol. 267, 2000, pages 159 - 173, XP004436101, DOI: doi:10.1006/viro.1999.0126
HEATH, G.S.; KING, D. P.; TURNER, J.L.E.; WAKELEY, P.R.; BANKS, M.: "Use of an internal standard in a TaqMano nested reverse transcription-polymerase chain reaction for the detection of bovine viral diarrhoea virus", VET. MICRO., vol. 96, 2003, pages 357 - 366, XP003000088, DOI: doi:10.1016/j.vetmic.2003.09.006
HIETALA, S.K.; CROSSLEY, B.M.: "Armored RNA as virus surrogate in a real-time reverse transcriptase PCR assay proficiency panel", J. CLIN. MICRO., vol. 44, 2006, pages 67 - 70
HOFFMAN ET AL.: "A review of RT-PCR technologies used in veterinary virology and disease control: Sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health", VETERINARY MICROBIOLOGY, vol. 139, 2009, pages 1 - 23, XP026642082, DOI: doi:10.1016/j.vetmic.2009.04.034
HOFFMANN, B; DEPNER, K.; SCHIRRMEIER, H.; BEER, M.: "A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses", J. VIROL. METHS., vol. 136, 2006, pages 200 - 209, XP025030292, DOI: doi:10.1016/j.jviromet.2006.05.020
KING ET AL: "Development of a novel recombinant encapsidated RNA particle: Evaluation as an internal control for diagnostic RT-PCR", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 146, no. 1-2, 3 November 2007 (2007-11-03), pages 218 - 225, XP022327158, ISSN: 0166-0934, DOI: DOI:10.1016/J.JVIROMET.2007.07.002 *
KING, D.P.; FERRIS, N.P.; SHAW, A.E.; REID, S.M.; HUTCHINGS, G.H.; GIUFFE, A.C.; ROBIDA, J.M.; CALLAHAN, J.D.; NELSON, W.M.; BECKH: "Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time RT-PCR assays", J. VET. DIAGN. INVEST., vol. 18, 2006, pages 92 - 96
KING, D.P.; MONTAGUE, N.; EBERT, K.; REID, S.M.; JUKES, J.P.; SCHADLICH, L.; BELSHAM, G.J.; LOMONOSSOFF, G.P.: "Development of a novel recombinant encapsidated RNA particle: Evaluation as an internal control for diagnostic RT-PCR", J.VIROL. METH., vol. 146, 2007, pages 218 - 225, XP022327158, DOI: doi:10.1016/j.jviromet.2007.07.002
KING, D.P.; REID S.M.; HUTCHINGS G.H.; GRIERSON, S.S.; WILKINSON, P.J.; DIXON, L.K.; BASTOS, A.D.S.; DREW, T.W.: "Development of a TaqMane PCR with internal amplification control for the detection of African swine fever virus", J. VIROL. METHS., vol. 107, 2003, pages 53 - 61
LIU, L.; CANIZARES, M.C.; MONGER, W.; PERRIN, Y.; TSAKIRIS, E.; PORTA, C.; SHARIAT, N.; NICHOLSON, L.; LOMONOSSOFF, G.P.: "Cowpea mosaic virus-based systems for the production of antigens and antibodies in plants", VACCINE, vol. 23, 2005, pages 1788 - 1792, XP004768055, DOI: doi:10.1016/j.vaccine.2004.11.006
LIU, L.; LOMONOSSOFF, G.P.: "Agroinfection as a rapid method for propagating cowpea mosaic virus-based constructs", J. VIROL. METHS., vol. 105, 2002, pages 343 - 348, XP009085158, DOI: doi:10.1016/S0166-0934(02)00121-0
MECHTCHERIAKOVA, I.A.; ELDAROV, M.A.; NICHOLSON, L.; SHANKS, M.; SKRYABIN, K. G.; LOMONOSSOFF, G. P.: "The use of viral vectors to produce hepatitis B virus core particles in plants", J. VIROL. METHS., vol. 131, 2006, pages 10 - 15, XP025030044, DOI: doi:10.1016/j.jviromet.2005.06.020
MONGER, W.; ALAMILLO, J.M.; SOLA, I.; PERRIN, Y.; BESTAGNO, M.; BURRONE, O.R.; SABELLA, P.; PLANA-DURAN, J.; ENJUANES, L.; GARCIA,: "An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus when supplied orally in crude plant extracts", PLANT BIOTECHNOL. J., vol. 4, 2006, pages 623 - 631
NAGEL, R.; ELLIOT, A.; MASEL, A.; BIRCH, R.G.; MANNERS, J.M.: "Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes", FEMS MICROBIOL. LETT., vol. 67, 1990, pages 325 - 328, XP023918298, DOI: doi:10.1111/j.1574-6968.1990.tb04041.x
PASLOSKE, BL.; WALKERPEACH, C.R.; OBERMOELLER, R.D.; WINKLER, M.; DUBOIS, D.B.: "Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards", J. CLIN. MICRO., vol. 36, 1998, pages 3590 - 3594
REID, S.M.; FERRIS, N.P.; HUTCHINGS, G.H.; KING, D.P.; ALEXANDERSEN, S.: "Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus", J. VIROL. METHS., vol. 116, 2004, pages 169 - 176
REID, S.M.; FERRIS, N.P.; HUTCHINGS, G.H.; ZHANG, Z.; BELSHAM, G.J.; ALEXANDERSEN, S.: "Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay", J. VIROL. METHS., vol. 105, 2002, pages 67 - 80, XP002244068, DOI: doi:10.1016/S0166-0934(02)00081-2
REID, S.M.; GRIERSON, S.S.; FERRIS, N.P.; HUTCHINGS, G.H.; ALEXANDERSEN, S.: "Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus", J. VIROL. METHS., vol. 107, 2003, pages 129 - 139
TAYLOR, K.M.; SPALL, V.E.; BUTLER, P.J.G.; LOMONOSSOFF, G.P.: "The cleavable carboxyl terminus of the small coat protein of cowpea mosaic virus is involved in RNA encapsidation", VIROLOGY, vol. 255, 1999, pages 129 - 137, XP004467113, DOI: doi:10.1006/viro.1998.9567
VAN KAMMEN, A.; DE JAGER, C.P.: "Cowpea mosaic virus", CMI/AAB DESCRIPTIONS OF PLANT VIRUSES, 1978
VAN LENT, J.; WELLINK, J.; GOLDBACH, R.: "Evidence for the ninvolvement of the 58K and 48K proteins in the intercellular movement of cowpea mosaic virus", J. GEN. VIROL., vol. 71, 1990, pages 219 - 223
WELLINK J.; VAN KAMMEN, A.: "Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins", J. GEN. VIROL., vol. 70, 1989, pages 2279 - 2286
WESTCOTT, D.G.; KING, D.P.; DREW, T.W.; NOWOTNY, N; KINDERMANN, J.; HANNANT, D.; BELÁK, S.; PATON, D.J.: "Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes", VET. RES., vol. 34, 2003, pages 165 - 17

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015118183A1 (fr) * 2014-02-10 2015-08-13 Amptec Gmbh Arn enveloppé
CN113025610A (zh) * 2019-12-09 2021-06-25 广西壮族自治区兽医研究所 可视化二重lamp鉴别禽白血病病毒和鸡传染性贫血病毒的方法
CN113025610B (zh) * 2019-12-09 2023-07-28 广西壮族自治区兽医研究所 可视化二重lamp鉴别禽白血病病毒和鸡传染性贫血病毒的方法

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