EP4565715A1 - Compositions, kits et procédés de détection de souches variantes du virus de la peste porcine africaine - Google Patents

Compositions, kits et procédés de détection de souches variantes du virus de la peste porcine africaine

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Publication number
EP4565715A1
EP4565715A1 EP23758107.9A EP23758107A EP4565715A1 EP 4565715 A1 EP4565715 A1 EP 4565715A1 EP 23758107 A EP23758107 A EP 23758107A EP 4565715 A1 EP4565715 A1 EP 4565715A1
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EP
European Patent Office
Prior art keywords
asfv
assay
variant
generic
sub
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP23758107.9A
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German (de)
English (en)
Inventor
Elise MARTIN
Sandrine MOINE
Aurore BLANC
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Life Technologies Corp
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Life Technologies Corp
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Publication of EP4565715A1 publication Critical patent/EP4565715A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • 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
    • 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
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present teachings relate to compositions, methods, systems, and kits for the detection of African swine fever virus (ASFV) in a test sample, and in particular, for distinguishing between wild/reference type ASFV and mutant/variant strains of ASFV.
  • ASFV African swine fever virus
  • ASFV African swine fever
  • ASF African swine fever
  • ASF is associated with hemorrhagic fever and high mortality rates in domestic pigs.
  • infected animals lose weight and often develop pneumonia, skin ulcers, and swollen joints.
  • Pregnant sows that contract ASF will often undergo spontaneous abortion, or the infection will lead to stillbirths
  • ASF thus represents a serious challenge to domestic pig operations in several regions of the world. Even in regions that have not yet experienced an ASF outbreak, such as the United States, the risk of transmission and outbreak remains present.
  • ASFV passes from a soft tick that infects several types of wild African swine, including giant forest hogs, warthogs, and bushpigs. Infection is generally asymptomatic in wild hosts.
  • ASF may be spread by infected ticks, but most transmission of concern is caused by transmission between pigs.
  • the virus may be transmitted through direct or indirect contact with infected pigs, their feces, or their body fluids. The virus also survives for multiple months or even years within pork products, so slaughtered pigs (from hunting or domestic production) can be a transmission vector.
  • Figure 1 illustrates a schematic of an ASFV genome to show example loci that can be targeted in order to detect ASFV and to distinguish between wild type and certain vaccine- associated variants.
  • Figure 2 illustrates an example of two separate process flows (i .e., methods) for using a combined ASFV assay that includes a generic ASFV sub-assay with an IPC and a variant ASFV sub-assay without an IPC.
  • Figure 3 illustrates an example of an external positive control (EPC) that may be utilized in conjunction with variant primer/probe sets (e.g., as in Table 1) in an assay for determining whether ASFV present in a sample is wild type or a variant.
  • EPC external positive control
  • Figure 1 illustrates a schematic of an ASFV genome to show example loci that can be targeted in order to detect ASFV and to distinguish between wild type and certain vaccine- associated variants.
  • a first target includes an MGF360 gene (e.g., the MGF360-14L gene), and a second target includes the CD2v gene.
  • MGF360 gene e.g., the MGF360-14L gene
  • CD2v gene e.g., the CD2v gene
  • a generic target may also be included.
  • Figure 1 shows an example of such a generic target as the p72 gene.
  • Assays disclosed herein are designed to target such genes to enable detection of ASFV and to determine whether detected ASFV is wild type oris likely to be a vaccine-associated variant.
  • detection of the generic target e.g., the p72 target
  • One or more of the other targets may additionally be analyzed to further characterize the detected ASFV. For example, if the first and second targets are detected, the assay result may be considered as positive for the wild type strain. If neither of the first or second targets are detected, but the generic target was detected, the assay result may be considered as positive for a double deletion, vaccine-associated variant. If one of the first or second targets is detected, but the other is not detected, the assay result may be considered as positive for another type of ASFV variant.
  • test sample for the assays described herein may include or be derived from a variety of sources, including blood, serum, saliva, tissues, feces, urine, or environmental samples exposed or suspected of potential exposure to infected animals.
  • Embodiments disclosed herein include primers and optionally probes useful for the detection of targeted ASFV loci in a sample associated with an animal. Such primers and probes can be used in singleplex or multiplex nucleic acid assays, as described in more detail below, for detection and identification of the targets in a sample.
  • the assays described herein demonstrate a high level of sensitivity, specificity, and accuracy.
  • the assay is designed to (I) detect the presence of ASFV in the sample, and (2) determine whether the ASFV is wild type ASFV or is a variant, such as a vaccine-associated variant.
  • assays are configured to detect an amplification product of the target regions by detecting a signal from a label (i.e., a detectable label) or other signal-generating process, where the signal indicates formation of the amplification product.
  • the label is attached to, or otherwise associated with, the corresponding forward primer and/or reverse primer used to generate the amplification product.
  • the label is attached to, or otherwise associated with, a probe configured to associate with a probe binding sequence within the target region.
  • the label is an optically detectable label.
  • the label may be detectable via non-optical means including electronically, electrically, or using NMR, sound, radioactivity, and the like.
  • the probes may be configured as TaqMan probes, which are known in the art and described in greater detail below. Such probes are able to hybridize to a target downstream from a primer such that exonuclease activity of the polymerase during subsequent primer extension separates a dye label from a quencher to increase the dye signal.
  • the assay is multiplex and includes differentially labelled probes.
  • a first probe targeted to a first sample target e.g., an MGF360 gene
  • a second probe targeted to a second sample target e.g., the CD2v gene
  • a separate probe is associated with the generic target (e.g., the p72 gene) and includes a third label different from both the first and second labels.
  • the generic target is assayed in a separate reaction volume from the first and/or second targets, and therefore does not require a label that is different from both the first and second probes.
  • a separate probe is associated with an internal positive control (TPC) and includes a fourth label different from the first, second, and/or third labels.
  • TPC internal positive control
  • the IPC is analyzed in a separate reaction volume from the first and/or second targets (e.g., with the generic target but not the first and second targets), and therefore does not require a label that is different from both the first and second probes.
  • Example primers and probes that may be used to detect the presence of the MGF360- 14L target and the CD2v target are provided below in Table 1.
  • the MGF360-14L probe is labelled with VIC
  • the CD2v probe is labelled with FAM.
  • these labels may be swapped, or other suitable labels, as known in the art and/or as described elsewhere herein, may be additionally or alternatively be utilized, including, but not limited to, JUN, ABY, Alexa Fluor dye labels (e.g., AF647 and AF676), and combinations thereof.
  • Assays may include the primer/probe sets for one or both targets shown in Table 1 to aid in determining whether detected ASFV is wild type or is a deletion variant type, such as a double deletion variant that would suggest it is a possible vaccine-associated variant.
  • one or both primer/probe sets shown in Table 1 may be combined with a primer/probe set configured to detect the presence of a generic ASFV target, such as the p72 region.
  • the primer/probe set for the generic ASFV target may be combined with one or both primer/probe sets of Table 1 in a multiplex arrangement or as a separate component intended for use in a separate reaction volume.
  • the tested sample includes wild type ASFV. Detection of neither of the MGF360-14L or CD2v targets (in conjunction with detection of a generic ASFV target such a p72) suggests that the tested sample includes a variant form of ASFV, such as a vaccine-associated variant. Detection of one of the MGF360-14L and CD2v targets suggests that the tested sample includes another variant form of ASFV.
  • Example assay kits can include any of the primers and/or probes described herein, including the primer/probe sets of Table 1.
  • an assay kit further comprises a master mix.
  • the primers and/or probes may be pre-mixed with and included as part of the master mix.
  • the master mix may include, for example, a polymerase, nucleotides, one or more buffers, or one or more salts to promote amplification of the target when the mixture and a sample combined therewith are exposed to amplification conditions.
  • the primer/probe sets of Table 1 are combined with a master mix such as the TaqPathTM ProAmpTM Master Mix (Thermo Fisher Scientific, Catalog No. A30865) in a container.
  • An assay kit as disclosed herein may also include an external positive control (EPC).
  • EPC may be provided in a container separate from the container holding the master mix and primer/probe sets.
  • An example EPC is described in greater detail below with reference to Figure 3 and Table 2.
  • the assay kit includes an internal positive control (1PC), either pre-mixed with the master mix or provided in a separate container.
  • the assay kit excludes an IPC and is instead designed for use with a separate ASFV assay that targets a generic ASFV locus.
  • the separate generic ASFV assay includes an IPC that can be leveraged to negate the need for a separate IPC within the variant ASFV assay.
  • An example of such a generic ASFV assay is the VetMAXTM African Swine Fever Virus Detection Kit (Applied Biosystems, Catalog No. A28809).
  • both assays may be referred to as a “combined ASFV assay” that includes both a “variant ASFV sub-assay” and a “generic ASFV sub-assay.”
  • both the variant ASFV sub-assay and the generic ASFV sub-assay include their own IPCs.
  • only one of the variant ASFV sub-assay or the generic ASFV subassay includes an IPC.
  • the variant ASFV sub-assay omits an IPC, and the process flow is instead utilized in a manner that leverages the IPC of the generic ASFV sub-assay.
  • Other embodiments may instead include an IPC for the variant ASFV sub-assay and not the generic ASFV sub-assay.
  • no IPC is included.
  • both the variant ASFV sub-assay and the generic ASFV subassay may be designed as duplex assays.
  • a first probe with a first label may be associated with the first target (e.g., the MGF360-14L gene), and a second probe with a second, different label may be associated with the second target (e g., the CD2v gene).
  • a first probe with a first label may be associated with the generic target (e.g., the p72 gene), and a second probe with a second, different label may be associated with the IPC.
  • the variant ASFV sub-assay may therefore be duplex with respect to two targeted loci where mutations/deletions are possible, whereas the generic ASFV sub-assay may be duplex with respect to the generic ASFV target and the IPC.
  • Figure 2 illustrates an example of two separate process flows (i.e., methods) for using a combined ASFV assay that includes a generic ASFV sub-assay with IPC and a variant ASFV sub-assay without IPC.
  • the “reagents” of the sub-assays refers to the respective master mixes of the sub-assays as described above (including respective primer/probe sets) in addition to any other desired components such as nuclease-free water, additional buffer, etcetera.
  • the test sample is mixed with the reagents of the generic ASFV sub-assay and the IPC in a first set of reaction volumes and with the reagents of the variant ASFV sub-assay in a second set of reaction volumes.
  • the amplification reactions of each set of reaction volumes are carried out together.
  • the two sub-assays may be carried out together by dividing the wells of a well-plate such that a first set of wells include the test sample, the reagents of the generic ASFV sub-assay, and the IPC, while a second set of wells include the test sample and the reagents of the variant ASFV sub-assay.
  • reaction volume arrangements may be utilized instead of or in addition to well plates. However, it is preferred that regardless of how the separate sets of reaction volumes are configured, the amplification reactions are carried out simultaneously or at least using the same instrument with substantially identical settings so that the IPC results of the generic ASFV reaction volumes remain applicable to the variant ASFV reaction volumes.
  • the generic ASFV sub-assay and the variant ASFV sub-assay are carried out sequentially.
  • the test sample is mixed with the reagents of the generic ASFV sub-assay and the IPC.
  • Amplification is then carried out. Only those wells in which the generic target (e.g., p72) is detected and in which the IPC is detected/validated are further analyzed using the variant ASFV sub-assay reagents to detect the presence of potential mutations/deletions (e.g., MGF360-14L and CD2v).
  • the first amplification reaction thus acts as a screening step to determine whether ASFV (in some form) is present before then further analyzing for the presence or absence of the variant-associated targets.
  • An assay kit as disclosed herein may also include an EPC.
  • the EPC may be provided in a container separate from the container holding the master mix and primer/probe sets.
  • an assay kit includes a first container comprising the primer/probe sets of Table I (or similar primer/probe sets for other suitable ASFV targets associated with a mutant/variant), and a second container comprising the EPC.
  • FIG. 3 illustrates an example of an EPC that may be utilized in conjunction with variant primer/probe sets (e.g., as in Table 1) in an assay for determining whether ASFV present in a sample is wild type ASFV or a variant ASFV.
  • the EPC is based on a GAPDH plasmid modified to replace the GAPDH primer and probe regions with ASFV primer and probe regions that correspond to the primer/probe sets of the kit.
  • the EPC plasmid is preferably designed such that the resulting amplicon length and distance between primers and probe substantially match the amplicon length and distance between primers and probe in the test sample reaction volumes.
  • the EPC may include multiple inserts (e.g., one for the MGF360-14L target and one for the CD2v target). Alternatively, multiple plasmids, each with a single target, may together be utilized as the EPC.
  • Table 2 lists example insert sequences for the MGF360-14L and CD2v targets that may be incorporated into a plasmid (e.g., a GAPDH plasmid as in Figure 3 or another suitable plasmid) to form an EPC suitable for use with an assay that includes the primer/probe sets of Table 1.
  • the inserts may both be added to the same plasmid or may be singly added to separate plasmids that are then combined.
  • Amplified products resulting from use of one or more embodiments described herein can be generated, detected, and/or analyzed on any suitable platform.
  • the nucleic acid targets may be single-stranded, double-stranded, or any other nucleic acid molecule of any size or conformation.
  • the amplification processes described herein can include PCR (see, e.g., U.S. Pat. No. 4,683,202).
  • the PCR is real time or quantitative PCR (qPCR).
  • the PCR is an end point PCR.
  • the PCR is digital PCR (dPCR).
  • Other amplification methods such as, e.g., loop-mediated isothermal amplification (“LAMP”), and other isothermal methods are also contemplated for use with the assay embodiments described herein.
  • LAMP loop-mediated isothermal amplification
  • certain qPCR assays can be plated into individual wells of a single array or multi-well plate, such as for example a TaqMan Array Card (see, e.g., Thermo Fisher Scientific, Waltham, MA; Catalog Nos. 4346800 and 4342265) or a MicroAmp multi-well (e.g., 96-well, 384-well) reaction plate (see, e.g., Thermo Fisher Scientific, Waltham, MA; Catalog Nos. 4346906, 4366932, 4306737, 4326659 and N8010560).
  • a TaqMan Array Card see, e.g., Thermo Fisher Scientific, Waltham, MA; Catalog Nos. 4346800 and 4342265
  • MicroAmp multi-well e.g., 96-well, 384-well
  • the different qPCR assays present in different wells of an array or plate can be dried or freeze-dried in situ and the array or plate can be stored or shipped prior to use.
  • the concepts described herein may be utilized in in situ hybridization applications not necessarily associated with PCR.
  • the primers described herein are used in nucleic acid assays at a concentration from about 100 nM to 1 mM (e.g., 300 nM, 400 nM, 500 nM, etc.), including all concentration amounts and ranges in between.
  • the probes described herein are used in a nucleic acid assay at a concentration from about 50 nM to 500 nM (e g., 75 nM, 125 nM, 250 nM, etc.), including all concentration amounts and ranges in between.
  • the primers and/or probes described herein may further comprise a fluorescent or other detectable label.
  • the primers and/or probes may further comprise a quencher and in other embodiments the probes may further comprise a minor groove binder (MGB) moiety.
  • Suitable fluorescent labels include but are not limited to 6FAM, ABY, VIC, JUN, and FAM.
  • Suitable quenchers include but are not limited to QSY (e.g., QSY7 and QSY21), BHQ (Black Hole Quencher) and DFQ (Dark Fluorescent Quencher).
  • primer and probe sequences described herein need not have 100% homology/identity to their targets to be effective, though in some embodiments, homology is substantially 100% or exactly 100%.
  • one or more of the disclosed primer and/or probe sequences have a homology to their respective target of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or up to substantially 100% or exactly 100%
  • primers and/or probes may include primers and/or probes each with different homologies to their respective targets, and the homologies may be, for example, within a range with endpoints defined by any two of the foregoing values.
  • different assay products can be independently detected or at least discriminated from each other.
  • different assay products may be distinguished optically (e.g., using optically different labels for each qPCR assay) or can be discriminated using some other suitable method, including as described in U.S. Patent Publication No. 2019/0002963, which is incorporated herein by reference in its entirety.
  • the reaction vessel or volume can optionally include a tube, channel, well, cavity, site or feature on a surface, or alternatively a droplet (e.g., a microdroplet or nanodroplet) that may be deposited onto a surface or into a surface well or cavity, or suspended within (or partially bounded by) a fluid stream.
  • the reaction volume includes one or more droplets arrayed on a surface or present in an emulsion.
  • the reaction volumes can optionally be formed by fusion of multiple pre-reaction volumes containing different components of an amplification reaction.
  • pre-reaction volumes containing one or more primers can be fused with pre-reaction volumes containing human nucleic acid samples and/or polymerase enzymes, nucleotides, and buffer.
  • a surface contains multiple grooves, channels, wells, cavities, sites, or features defining a reaction volume containing one or more amplification reagents (e.g., primers, probes, buffer, polymerase, nucleotides, and the like).
  • the reaction volume within the selected tubes, grooves, channels, wells, cavities, sites, or features contains only a single forward primer sequence and a single reverse primer sequence.
  • one or more probe sequences are also included in the singleplex reaction volume.
  • Assays described herein may utilize TaqMan probes.
  • TaqMan-based assays are typically carried out by performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5'-to-3 ' nuclease activity.
  • the probe typically includes a detectable label (e.g., a fluorescent reporter molecule) and a quencher molecule capable of quenching the fluorescence of the reporter molecule.
  • the detectable label and quencher molecule are part of a single probe.
  • the polymerase digests the probe to separate the detectable label from the quencher molecule.
  • the detectable label is monitored during the reaction, where detection of the label corresponds to the occurrence of nucleic acid amplification (e.g., the higher the signal the greater the amount of amplification).
  • detection of the label corresponds to the occurrence of nucleic acid amplification (e.g., the higher the signal the greater the amount of amplification).
  • Variations of TaqMan assays are known in the art and would be suitable for use in the methods described herein.
  • primers can be labeled and used to both generate amplicons and to detect the presence (or concentration) of amplicons generated in the reaction, and such may be used in addition to or as an alternative to labeled probes described herein.
  • primers may be labeled and utilized as described in Nazarenko et al. (Nucleic Acids Res. 2002 May 1 ; 30(9): e37), Hayashi et al. (Nucleic Acids Res. 1989 May 11; 17(9): 3605), and/or Neilan et al. (Nucleic Acids Res. Vol. 25, Issue 14, 1 July 1997, pp. 2938-39).
  • Those of skill in the art will also understand and be capable of utilizing the PCR processes (and associated probe and primer design techniques) described in Zhu et al. (Biotechniques. 2020 Jul: 10.2I44/btn-2020-0057).
  • intercalating labels can be used such as ethidium bromide, SYBR Green I, SYBR GreenER, and PicoGreen (Life Technologies Corp., Carlsbad, CA), thereby allowing visualization in real-time, or end point, of an amplification product in the absence of a detector probe.
  • real-time visualization may include both an intercalating detector probe and a sequence-based detector probe.
  • the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction.
  • probes may further comprise various modifications such as a minor groove binder to further provide desirable thermodynamic characteristics.
  • the amplicon is labeled by incorporation of or hybridization to labeled primer. In some embodiments, the amplicon is labeled by hybridization to a labeled probe. In some embodiments, the amplicon is labeled by binding of a DNA-binding dye. In some embodiments, the dye may be a single-strand DNA binding dye. In other embodiments, the dye may be a double-stranded DNA binding dye. In other embodiments, the amplicon is labeled via polymerization or incorporation of labeled nucleotides in a template-dependent (or templateindependent) polymerization reaction.
  • the labeled nucleotide can be added after amplifying is completed.
  • the labeled amplicon (or labeled derivative thereof) can be detected using any suitable method such as, for example, electrophoresis, hybridization-based detection (e.g., microarray, molecular beacons, and the like), chromatography, NMR, and the like.
  • the labeled amplicon is detected using capillary electrophoresis.
  • the labeled amplicon is detected using qPCR.
  • a plurality of different amplicons is formed, and optionally labeled, within a single reaction volume via a single amplification reaction.
  • a multiplex reaction e.g., 2- plex, 3-plex, 4-plex, 5-plex, 6-plex
  • a single tube or reaction vessel e.g., “singletube” or “ I -tube” or “single-vessel” reaction
  • the plurality of amplicons can be differentially labeled.
  • each of the plurality of amplicons produced during amplification is labeled with a different label.
  • the nucleic acid amplification assays as described herein are performed using a Real-time PCR (qPCR) instrument, including for example a QuantStudio Real- Time PCR system, such as the QuantStudio 5 RealTime PCR System (QS5), QuantStudio 7 RealTime PCR System (QS7), and/or QuantStudio 12K Flex System (QS12K), or a 7500 Real- Time PCR system, such as the 7500 Fast Dx system, from Thermo Fisher Scientific.
  • qPCR Real-time PCR
  • embodiments described herein may also include properties and/or features (e.g., ingredients, components, members, elements, parts, and/or portions) described in one or more separate embodiments and are not necessarily limited strictly to the features expressly described for that particular embodiment. Accordingly, the various features of a given embodiment can be combined with and/or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include such features.

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Abstract

L'invention concerne des compositions, des procédés, des systèmes et des kits pour la détection du virus de la peste porcine africaine (ASFV) dans un échantillon d'essai, et en particulier pour distinguer entre des souches ASFV de type sauvage/référence et des souches mutantes/variantes d'ASFV. Un dosage d'ASFV variant comprend un premier ensemble d'amorces et une première sonde qui correspondent à une première cible d'ASFV, et un deuxième ensemble d'amorces et une deuxième sonde qui correspondent à une deuxième cible d'ASFV. La première et la deuxième sonde sont marquées différemment. La première cible ASFV est un gène MGF360 et la seconde cible ASFV est le gène CD2v. L'absence de ces cibles, associée à une détermination positive d'une autre cible générique du virus de la peste porcine africaine, telle que le gène p72, indique qu'il s'agit d'une souche variante d'ASFV associée à un vaccin.
EP23758107.9A 2022-08-05 2023-07-28 Compositions, kits et procédés de détection de souches variantes du virus de la peste porcine africaine Pending EP4565715A1 (fr)

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CN118755879B (zh) * 2024-06-07 2025-08-05 中国兽医药品监察所 三重荧光pcr鉴别诊断非洲猪瘟不同毒株三种基因的检测方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
WO2019006023A1 (fr) 2017-06-28 2019-01-03 ChromaCode, Inc. Mesures fluorimétriques multiplexées avec systèmes de pcr en gouttelettes
CN110878377B (zh) * 2019-11-06 2023-08-22 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 非洲猪瘟病毒强弱毒荧光定量pcr鉴别诊断试剂盒
CN111074000B (zh) * 2019-11-18 2023-08-22 华南农业大学 一种区分asfv野毒株与双基因缺失株的三重荧光定量pcr检测材料及试剂盒
CN112831598B (zh) * 2019-11-22 2022-12-20 洛阳普泰生物技术有限公司 用于非洲猪瘟病毒鉴别检验的实时荧光pcr扩增引物对、探针引物、及制备的试剂盒
CN113122655A (zh) * 2019-12-30 2021-07-16 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 非洲猪瘟病毒EP402R基因TaqMan荧光定量PCR检测方法
CN112094950A (zh) * 2020-10-15 2020-12-18 杭州博日科技股份有限公司 用于检测非洲猪瘟病毒野毒株和基因缺失株的引物组、试剂盒、方法和应用
CN114395643A (zh) * 2022-02-21 2022-04-26 华南农业大学 非洲猪瘟病毒的双通道数字pcr检测试剂盒及方法
CN114774581B (zh) * 2022-03-11 2024-07-12 山东舜丰生物科技有限公司 一种用于检测非洲猪瘟病毒的pcr引物和探针组合物

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