WO2004058300A1 - Sequences diagnostic for foot and mouth disease - Google Patents
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- WO2004058300A1 WO2004058300A1 PCT/US2003/041808 US0341808W WO2004058300A1 WO 2004058300 A1 WO2004058300 A1 WO 2004058300A1 US 0341808 W US0341808 W US 0341808W WO 2004058300 A1 WO2004058300 A1 WO 2004058300A1
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- 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
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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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
- 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
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
Definitions
- FMD is one of the most feared reportable diseases known in North America. Disease caused by FMDV is devastating to farm animals and can have a major economic impact on countries producing cloven-hoofed animals (cattle, pigs, sheep, goats and camelids) or their products. Clearly, new and more sensitive assays for the detection of this disease are needed. A variety of methods for the detection of FMDV have been developed. These fall into three general categories: 1) detection of FMDV peptides; 2) detection of FMDV generated antibodies; and 3) detection of FMDV genetic material.
- SEQ ID NO: 17 is the nucleotide sequence of a 3' Reverse diagnostic primer, P33-4, which binds to 4086-4108 bp of GenBank AF308157.
- SEQ ID NO:18 is the nucleotide sequence of a 3' Reverse diagnostic primer, P33+, which binds to 4083-4111 bp of GenBank AF308157.
- SEQ ID NO: 19 is the nucleotide sequence of a 3' Reverse diagnostic primer, LJS1 , which binds to 4460-4489 bp of GenBank AF308157.
- isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
- polynucleotide polynucleotide sequence
- nucleic acid sequence and “nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like.
- amplification product refers to nucleic acid fragments produced during a primer-directed amplification reaction.
- Typical methods of primer-directed amplification include polymerase chain reaction (PCR), reverse transcription followed by PCR (RT-PCR), ligase chain reaction (LCR) or strand displacement amplification (SDA).
- PCR polymerase chain reaction
- RT-PCR reverse transcription followed by PCR
- LCR ligase chain reaction
- SDA strand displacement amplification
- the replication composition may comprise the components for nucleic acid replication, for example: nucleotide triphosphates, two (or more) primers with appropriate sequences, DNA or RNA polymerase, buffers, solutes and proteins.
- RNA-polymerase promoter systems (T7 RNA polymerase) are also contemplated.
- probe refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest.
- non- participatory will refer to the lack of participation of a probe or primer in a reaction for the amplification of a nucleic acid molecule.
- a non-participatory probe or primer is one that will not serve as a substrate for, or be extended by, a DNA or RNA polymerase.
- a "non-participatory probe” is inherently incapable of being chain extended by a polymerase. It may or may not have a replication inhibitor moiety.
- a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength.
- Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.
- low stringency hybridization conditions corresponding to a Tm of 55°
- 5X SSC 0.1% SDS, 0.25% milk, and no formamide
- 30% formamide 5X SSC, 0.5% SDS
- Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5X or 6X SSC.
- Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
- the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.
- a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
- Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
- Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.
- Expression cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
- sequence analysis software refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences.
- Sequence analysis software may be commercially available or independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wl), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990), DNASTAR (DNASTAR, Inc., Madison, Wl), and Vector NTi version 7.0.
- Foot-and-mouth disease virus O genomic RNA, isolate 01 Campos, complete genome (Accession No. AJ320488); Pereda,A.J., et al. Arch. Virol. 147 (11 ): 2225-2230 (2002); 2. Foot-and-mouth disease virus SAT 2, complete genome (Accession No. NC003992);
- Foot-and-mouth disease virus O complete genome (Accession No. AF308157); Beard, C.W. and Mason, P.W. J. Virol. 74 (2): 987- 991 (2000)).
- primers are broadly useful to detect FDMV infections across a plurality of serotypes and variations and in FMDV infections
- SEQ ID NOs: 16-20 may be used in a variety of formats for the detection of FMDV. Most preferred are primer-directed amplification methods and nucleic acid hybridization methods.
- the sample and methods of collecting the sample may include, but are not limited to: swabs from oral and nasal cavities, body fluids (e.g., blood, blood serum, urine, fecal material, saliva, cerebrospinal fluid, lymph fluid, amniotic fluid, peritoneal fluid), tissues (e.g., muscle, skin) or bone samples. Additionally, air and soil samples may be used.
- body fluids e.g., blood, blood serum, urine, fecal material, saliva, cerebrospinal fluid, lymph fluid, amniotic fluid, peritoneal fluid
- tissues e.g., muscle, skin
- air and soil samples may be used.
- SEQ ID NOs: 16-20 may be used as primers for use in primer-directed nucleic acid amplification for the detection of the presence of FMDV.
- primer-directed nucleic acid amplification methods include thermal cycling methods (e.g., PCR, RT-PCR, and LCR), as well as isothermal methods and strand displacement amplification (SDA).
- the primers typically have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid.
- Methods of PCR primer design are common and well-known in the art (Thein and Wallace, "The use of oligonucleotide as specific hybridization probes in the Diagnosis of Genetic Disorders", In Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50; IRL: Herndon, VA; and Rychlik, W. (1993) In White, B. A. (ed.), Methods in Molecular Biology. Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, NJ).
- DNTP 200 or 250 ⁇ m Forward primer e.g. P2Fwd-10 600 nM
- Reverse primer (e.g. P33-4) 2 ⁇ M
- Reverse Primer (e.g. P33-4) 2.4 ⁇ M
- Preferred RT-PCR cycling conditions are:
- Primer-directed amplification products can be analyzed using various methods.
- Homogenous detection refers to a preferred method for the detection of amplification products where no separation (such as by gel electrophoresis) of amplification products from template or primers is necessary. Homogeneous detection is typically accomplished by measuring the level of fluorescence of the reaction mixture in the presence of a fluorescent dye.
- DNA melting curve analysis is used to carry out homogenous detection, particularly with the BAX® System hardware and reagent tablets from Qualicon Inc.
- BAX® System hardware and reagent tablets from Qualicon Inc.
- the details of the system are given in U.S. Patent No. 6,312,930 and PCT Publication Nos. WO 97/11197 and WO 00/66777, each of which is hereby incorporated by reference in its entirety.
- dsDNA double stranded nucleic acid molecule
- target amplicon target amplification product
- the two strands of a dsDNA separate or melt, when the temperature is higher than its melting temperature.
- Melting of a dsDNA molecule is a process, and under a given solution condition, melting starts at a temperature (designated TM S hereinafter), and completes at another temperature (designated TME hereinafter).
- TM S melting start at a temperature
- TME completes at another temperature
- T m designates the temperature at which melting is 50% complete.
- a typical PCR cycle involves a denaturing phase where the target dsDNA is melted, a primer annealing phase where the temperature optimal for the primers to bind to the now-single-stranded target, and a chain elongation phase (at a temperature TE) where the temperature is optimal for DNA polymerase to function.
- TMS should be higher than TE, and TME should be lower (often substantially lower) than the temperature at which the DNA polymerase is heat-inactivated.
- Melting characteristics are effected by the intrinsic properties of a given dsDNA molecule, such as deoxynucleotide composition and the length of the dsDNA.
- Intercalating dyes will bind to double stranded DNA.
- the dye/dsDNA complex will fluoresce when exposed to the appropriate excitation wavelength of light, which is dye dependent, and the intensity of the fluorescence may be proportionate to concentration of the dsDNA.
- Methods taking advantage of the use of DNA intercalating dyes to detect and quantify dsDNA are known in the art. Many dyes are known and used in the art for these purposes. The instant methods also take advantage of such relationship.
- Such dyes includes intercalating dyes.
- examples of such dyes include, but are not limited to, SYBR Green-I®, ethidium bromide, propidium iodide, TOTO®-1 ⁇ Quinolinium, 1-1'-[1 ,3-propanediylbis [(dimethyliminio) -3,1-propanediyl]]bis[4-[(3- methyI-2(3H)-benzothiazolylidene) methyl]]-, tetraiodide ⁇ , and YoPro® ⁇ Quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3- (trimethylammonio)propyl]-,diiodide ⁇ .
- Most preferred for the instant invention is a non-asymmetrical cyanide dye such as SYBR Green-I®, manufactured by Molecular Probes, Inc. (Eugene, OR).
- Melting curve analysis is achieved by monitoring the change in fluorescence while the temperature is increased. When the temperature reaches the TMS specific for the target amplicon, the dsDNA begins to denature. When the dsDNA denatures, the intercalating dye dissociates from the DNA and fluorescence decreases.
- Mathematical analysis of the negative of the change of the log of fluorescence divided by the change in temperature plotted against the temperature results in the graphical peak known as a melting curve (See Figure 6, which illustrates melting curve analysis in general).
- a positive detection for FMDV results in the appearance of a melting curve peak as follows:
- the instant homogenous detection method can be used to detect and quantify target dsDNAs, from which the presence and level of target organisms can be determined. This method is very specific and sensitive.
- Homogenous detection may be employed to carry out "real-time” primer-directed nucleic acid amplifications, using primer pairs of the instant invention (e.g., “real-time” PCR and “real-time” RT-PCR).
- PCR detection methods include standard non-denaturing gel electrophoresis (e.g., acrylamide or agarose), denaturing gradient gel electrophoresis, and temperature gradient gel electrophoresis.
- standard non-denaturing gel electrophoresis is a simple and quick method of PCR detection, but may not be suitable for all applications.
- DGGE exploits the fact that otherwise identical DNA molecules having the same length and DNA sequence, with the exception of only one nucleotide within a specific denaturing domain, will denature at different temperatures or Tm.
- ds double-stranded
- the dsDNA fragment will travel faster than a denatured single- stranded (ss) DNA fragment, since the branched structure of the single- stranded moiety of the molecule becomes entangled in the gel matrix.
- the ds DNA fragment will completely dissociate and mobility of the molecule through the gel is retarded at the denaturant concentration at which the particular low denaturing domains of the DNA strand dissociate.
- the electrophoresis is conducted at a constant temperature (around 60°C) and chemical denaturants are used at concentrations that will result in 100% of the DNA molecules being denatured (i.e., 40% formamide and 7M urea).
- This variable denaturing gradient is created using a gradient maker, such that the composition of each DGGE gel gradually changes from 0% denaturant up to 100% denaturant.
- gradients containing a reduced range of denaturant e.g., 35% to 60%
- DGGE Temperature Gradient Gel Electrophoresis
- TGGE Temperature Gradient Gel Electrophoresis
- This method makes use of a temperature gradient to induce the conformational change of dsDNA to ssDNA to separate fragments of equal size with different sequences.
- DNA fragments with different nucleotide sequences will become immobile at different positions in the gel.
- Variations in primer design can be used to advantage in increasing the usefulness of DGGE for characterization and identification of the PCR products. These methods and principles of using primer design variations are described in PCR Technology Principles and Applications, Henry A. Erlich Ed., M. Stockton Press, NY, pages 71 to 88 (1988).
- the BAX ® System DuPont Qualicon, Wilmington, DE
- melting curve analysis are used.
- replication composition Any suitable nucleic acid replication composition in any format can be used.
- replication composition is in liquid form, suitable buffers known in the art may be used (Sambrook, J. et al. 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
- suitable buffers known in the art may be used (Sambrook, J. et al. 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
- typical tabletization reagents may be included such as stabilizers and binding agents.
- Preferred tabletization technology is set forth in U.S. Patent Nos. 4,762,857 and 4,678,812, each of which is hereby incorporated by reference in its entirety.
- a preferred kit for detection of FMDV comprises (a) at least one pair of PCR primers selected from the group consisting of (i) SEQ ID NOs:16 and 17, (ii) SEQ ID NOs:16 and 18, (iii) SEQ ID NOs:16 and 19; and (iv) SEQ ID NOs:16 and 20; (b) thermostable DNA polymerase; and (c) reverse transcriptase.
- a preferred tablet comprises (a) at least one pair of PCR primers selected from the group consisting of (i) SEQ ID NOs:16 and 17, (ii) SEQ ID NOs:16 and 18, (iii) SEQ ID NOs:16 and 19; and (iv) SEQ ID NOs:16 and 20; (b) thermostable DNA polymerase; and (c) reverse transcriptase.
- a kit for detection of FMDV comprises the foregoing preferred tablet.
- Probes particularly useful in nucleic acid hybridization methods are any of SEQ ID NOs: 16-20 or sequences derived therefrom.
- the basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing FMDV, and a specific hybridization method.
- Probes are single stranded nucleic acid sequences which are complementary to the nucleic acid sequences to be detected.
- Probes are "hybridizable" to the nucleic acid sequence to be detected.
- the probe length can vary from as few as 5 bases to the full length of the FMDV diagnostic sequence and will depend upon the specific test to be done. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected.
- a probe may be composed of either RNA or DNA.
- the form of the nucleic acid probe may be a marked single stranded molecule of just one polarity or a marked single stranded molecule having both polarities present.
- the form of the probe like its length, will be determined by the type of hybridization test to be done.
- the sample may or may not contain the FMDV.
- the sample may take a variety of forms, however will generally be extracted from an animal, environmental or food source suspected of coming in contact with the FMDV.
- hybridization methods are well defined.
- the probe and sample must be mixed under conditions which will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions.
- the probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur.
- the concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed.
- hybridization assays may be conducted directly on cell lysates, without the need to extract the nucleic acids. This eliminates several steps from the sample-handling process and speeds up the assay.
- Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate, among others.
- the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
- a variety of methods are known to one of skill in the art (e.g., phenol-chloroform extraction, IsoQuick extraction (MicroProbe Corp., Bothell, WA), and others).
- Pre-hybridization purification is particularly useful for standard filter hybridization assays.
- purification facilitates measures to increase the assay sensitivity by incorporating in vitro RNA amplification methods such as self-sustained sequence replication (see for example Fahy et al., In PCR Methods and Applications, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1991), pp. 25-33) or reverse transcriptase PCR (Kawasaki, In PCR Protocols: A Guide to Methods and Applications, M. A. Innis et al., Eds., (1990), pp. 21-27).
- RNA or DNA can be detected by any of a variety of methods. However, the most useful embodiments have at least some characteristics of speed, convenience, sensitivity, and specificity.
- Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent.
- a common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1 M buffers, such as sodium citrate, Tris-HCI, PIPES or HEPES (pH range about 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about
- unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine.
- volume exclusion agents which include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate), and anionic saccharidic polymers (e.g., dextran sulfate).
- polar water-soluble or swellable agents e.g., polyethylene glycol
- anionic polymers e.g., polyacrylate or polymethylacrylate
- anionic saccharidic polymers e.g., dextran sulfate
- Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non- denaturing conditions.
- a primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the DNA sequence.
- the sandwich assay may be encompassed in an assay kit.
- This kit would include a first component for the collection of samples from an animal suspected of having contracted the FMDV and buffers for the disbursement and lysis of the sample.
- a second component would include media in either dry or liquid form for the hybridization of target and probe polynucleotides, as well as for the removal of undesirable and nonduplexed forms by washing.
- a third component includes a solid support (dipstick) upon which is fixed (or to which is conjugated) unlabeled nucleic acid probe(s) that is (are) complementary to a part of the FMDV genome.
- a fourth component would contain labeled probe that is complementary to a second and different region of the same DNA strand to which the immobilized, unlabeled nucleic acid probe of the third component is hybridized.
- SEQ ID NOs: 16-20 or derivations thereof may be used as 3' blocked detection probes in either a homogeneous or heterogeneous assay format.
- a probe generated from these sequences may be 3' blocked or non-participatory and will not be extended by, or participate in, a nucleic acid amplification reaction.
- the probe incorporates a label that can serve as a reactive ligand that acts as a point of attachment for the immobilization of the probe/analyte hybrid or as a reporter to produce detectable signal.
- genomic or cDNA isolated from a sample suspected of harboring the FMDV is amplified by standard primer-directed amplification protocols in the presence of an excess of the 3' blocked detection probe to produce amplification products. Because the probe is 3' blocked, it does not participate or interfere with the amplification of the target. After the final amplification cycle, the detection probe anneals to the relevant portion of the amplified DNA and the annealed complex is then captured on a support through the reactive ligand. In some instances it is desirable to incorporate a ligand labeled dNTP, with the label probe in the replication composition to facilitate immobilization of the RT-PCR reaction product on a support and then detection of the immobilized product by means of the labeled probe reagent.
- cordycepin Since the cordycepin will be attached to the 3' terminal end of the probe, the synthesis is initiated from a cordycepin covalently attached to CPG, 5-dimethoxytrityl- N-benzoyl-3-deoxyadenosine (cordycepin), 2-succinoyl-long chain alkylamino-CPG (Glen Research, Sterling, VA). The dimethoxytrityl group is removed and the initiation of the chain synthesis starts at the deprotected 5' hydroxyl group of the solid phase cordycepin. After the synthesis is complete, the oligonucleotide probe is cleaved off the solid support leaving a free 2' hydroxyl group on the 3'-terminally attached cordycepin.
- reagents can also be attached to the 3' terminus during the synthesis of the non-participatory probe to serve as replication inhibitors. These include, but are not limited to: other 3-deoxyribonucleotides, biotin, dinitrophenol, fluorescein, and digoxigenin. Each of these reagents are also derivatized on CPG supports (Glen Research; Clonetech Laboratories, Palo Alto, CA).
- test kits and reagents were purchased from the following vendors: pCR4-TOPO vector (Invitrogen Life Technologies, Catalog #45-0030); Qiagen QIAquick PCR Purification Kit (Qiagen, Catalog #28104); Qiagen Rneasy Mini Kit (Catalog #74106); Qiagen QIAprep Spin Mini Prep Kit (Catalog # 27106); RNA Transcription kit (Stratagene, Catalog #200340, Cedar Creek, TX); and TOPO TA Cloning Kit Dual Promoter (Invitrogen Life Technologies, Catalog #45-0640).
- oligonucleotide primers and linkers were synthesized by Sigma Genosys Company, The Woodlands, TX. Polymerase chain reactions and RNA quantitations were performed using a PTC-225 Peltier Thermal Cycler (MJ Research Waltham, MA) and GeneQuant pro (Catalog #80-2110-98; Amersham Pharmacia Biotech, Cambridge, England).
- GCG Genetics Computer Group Inc.
- GCG program “Pileup” was used the gap creation default value of 12, and the gap extension default value of 4 were used.
- CGC “Gap” or “Bestfit” programs were used the default gap creation penalty of 50 and the default gap extension penalty of 3 were used. In any case where GCG program parameters were not prompted for, in these or any other GCG program, default values were used.
- a synthetic piece of a foot and mouth virus (FMDV) RNA serotype O (GenBank Accession Number AF308157; Beard, C.W. and Mason, P.W., J. Virology 74(2): 987-991 (2000)) was constructed from base 3800 to 4290.
- the synthetic FMD target was constructed using 13 total DNA linkers (SEQ ID NOs: 1-13) comprising both top and bottom strands ( Figure 1 ). Notl and EcoRI sites were added to the sequence of synthetic DNA target to facilitate directional cloning of the construct behind the T7 promoter in the pCR4-TOPO vector. Linkers were kinased, ligated and PCR amplified using primers
- Amplicon 5' and Amplicon 3' (SEQ ID NOs: 14 and 15, respectively) in accordance to published protocols with modifications (Maniatis, supra, pp 5.68-5.69, 1.68-1.69, 14.2-14.19).
- linkers SEQ ID NOs: 1-13 were diluted with DEPC treated water to 25 pmoles/ ⁇ L. Linkers (25 pmoles of each) were combined in one tube. To this tube 10 ⁇ L of 10x T4 Kinase buffer, 100 Units of T4 Kinase, 1 mM TP and DEPC water to 100 ⁇ L final volume was added. The reaction was incubated for 30 min at 37°C. The kinased linkers mix was heated at 95°C for 20 min in a heat , block to inactivate the kinase and melt all the linkers. After the 20 min the heat block was turned off and allowed to cool, thereby facilitating proper linker annealing.
- the ligation reaction was set-up as follows: in a total volume of 100 ⁇ l, 85 ⁇ L of the kinased- annealed linkers, 10 ⁇ L of 10X ligase buffer, and 50 Units of Ligase were added. The reaction proceeded for 30 min at room temperature or overnight at 14°C. Following ligation, the product was amplified by PCR to add restriction sites (if necessary) and to bulk up the quantity of product available for subsequent cloning.
- the PCR product produced above was cloned using topoisomerase-cloning technology (TOPO) developed by Invitrogen.
- TOPO topoisomerase-cloning technology
- the TOPO TA Cloning Dual Promoter Kit was used for the initial cloning of the synthetic FMD piece.
- Putative clones were transformed into competent E. coli provided by the Invitrogen kit (Top10F').
- E. coli harboring vectors (with or without inserts) were selected for on LB media containing 50 - 100 ⁇ g/ml ampicillian or carbenicillian for vector selection.
- Positive clones, containing the insert were determined by growing up individual colonies in 4 ml of LB broth supplemented with 100 ⁇ g/ml ampicillian overnight at 37°C with 230 rpm shaking.
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Priority Applications (5)
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US10/538,590 US7790876B2 (en) | 2002-12-20 | 2003-12-19 | Sequences diagnostic for foot and mouth disease |
EP03800441A EP1583556B1 (en) | 2002-12-20 | 2003-12-19 | Sequences diagnostic for foot and mouth disease |
BR0316917-0A BR0316917A (en) | 2002-12-20 | 2003-12-19 | Method for detecting fmdv, isolated polynucleotide, fmdv detection kits, replication composition and tablet |
DE60323153T DE60323153D1 (en) | 2002-12-20 | 2003-12-19 | DIAGNOSTIC SEQUENCES FOR MALE AND CLAUSE DISEASE |
AU2003300192A AU2003300192A1 (en) | 2002-12-20 | 2003-12-19 | Sequences diagnostic for foot and mouth disease |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1767657A1 (en) * | 2005-09-21 | 2007-03-28 | Fair Isaac Corporation | Detecting foot-and-mouth disease virus |
CN1308461C (en) * | 2004-12-24 | 2007-04-04 | 武汉大学 | Fluorescence quantitative PCR kit for detecting virus of aftosa and application |
CN105695628A (en) * | 2016-03-07 | 2016-06-22 | 华南农业大学 | HRM detecting primers and method for distinguishing foot-mouth disease virus and Seneca Valley virus |
CN107326100A (en) * | 2017-07-17 | 2017-11-07 | 河南省动物疫病预防控制中心 | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus |
US9944975B2 (en) | 2015-09-03 | 2018-04-17 | Abbott Molecular Inc. | Hybridization buffers |
CN108467903A (en) * | 2018-05-02 | 2018-08-31 | 郭庆君 | A kind of primer sets and kit for A types and O-shaped foot and mouth disease virus antidiastole |
US10457981B2 (en) | 2016-01-08 | 2019-10-29 | Abbott Molecular Inc. | Hybridization buffers |
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US7148343B2 (en) * | 2001-10-12 | 2006-12-12 | Gentra Systems, Inc. | Compositions and methods for using a solid support to purify RNA |
CA2586532C (en) | 2004-11-05 | 2014-03-25 | Qiagen North American Holdings, Inc. | Compositions and methods for purifying nucleic acids from stabilization reagents |
CN101724711B (en) * | 2008-10-30 | 2011-12-07 | 中国检验检疫科学研究院 | Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection |
CN114164299A (en) * | 2021-05-26 | 2022-03-11 | 郑州中道生物技术有限公司 | Foot-and-mouth disease (OAA 1) triple fluorescence RT-PCR detection kit |
Citations (1)
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WO2002095074A1 (en) * | 2001-05-18 | 2002-11-28 | Tetracore, Inc. | Foot and mouth disease virus diagnostic and methods |
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JP2813768B2 (en) | 1995-05-24 | 1998-10-22 | 農林水産省家畜衛生試験場長 | Foot-and-mouth disease diagnostic peptide and foot-and-mouth disease diagnostic antigen containing the peptide |
US6048538A (en) | 1997-10-03 | 2000-04-11 | United Biomedical, Inc. | Peptides derived from the non-structural proteins of foot and mouth disease virus as diagnostic reagents |
-
2003
- 2003-12-19 DE DE60323153T patent/DE60323153D1/en not_active Expired - Lifetime
- 2003-12-19 US US10/538,590 patent/US7790876B2/en not_active Expired - Fee Related
- 2003-12-19 EP EP03800441A patent/EP1583556B1/en not_active Expired - Fee Related
- 2003-12-19 AU AU2003300192A patent/AU2003300192A1/en not_active Abandoned
- 2003-12-19 BR BR0316917-0A patent/BR0316917A/en not_active Application Discontinuation
- 2003-12-19 WO PCT/US2003/041808 patent/WO2004058300A1/en active Application Filing
Patent Citations (1)
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WO2002095074A1 (en) * | 2001-05-18 | 2002-11-28 | Tetracore, Inc. | Foot and mouth disease virus diagnostic and methods |
Non-Patent Citations (3)
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BARANOWSKI E. ET AL.: "Multiple virulence determinants of foot-and-mouth disease virus in cell culture", J. OF VIROLOGY, vol. 72, no. 8, August 1998 (1998-08-01), pages 6362 - 6372, XP002977594 * |
PATTNAIK B. ET AL.: "Evaluation of primers for PCR amplification of RNA polymerase gene sequences of foot-and-mouth disease virus", ACTA VIROLOGICA, vol. 41, 1997, pages 333 - 336, XP002977593 * |
See also references of EP1583556A4 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1308461C (en) * | 2004-12-24 | 2007-04-04 | 武汉大学 | Fluorescence quantitative PCR kit for detecting virus of aftosa and application |
EP1767657A1 (en) * | 2005-09-21 | 2007-03-28 | Fair Isaac Corporation | Detecting foot-and-mouth disease virus |
US9944975B2 (en) | 2015-09-03 | 2018-04-17 | Abbott Molecular Inc. | Hybridization buffers |
US10457981B2 (en) | 2016-01-08 | 2019-10-29 | Abbott Molecular Inc. | Hybridization buffers |
CN105695628A (en) * | 2016-03-07 | 2016-06-22 | 华南农业大学 | HRM detecting primers and method for distinguishing foot-mouth disease virus and Seneca Valley virus |
CN105695628B (en) * | 2016-03-07 | 2019-02-01 | 华南农业大学 | A kind of HRM detection primer and method identifying swine foot-and-mouth disease virus and pig Sai Neijia paddy virus |
CN107326100A (en) * | 2017-07-17 | 2017-11-07 | 河南省动物疫病预防控制中心 | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus |
CN108467903A (en) * | 2018-05-02 | 2018-08-31 | 郭庆君 | A kind of primer sets and kit for A types and O-shaped foot and mouth disease virus antidiastole |
Also Published As
Publication number | Publication date |
---|---|
BR0316917A (en) | 2005-10-18 |
EP1583556B1 (en) | 2008-08-20 |
EP1583556A1 (en) | 2005-10-12 |
US7790876B2 (en) | 2010-09-07 |
US20080032285A1 (en) | 2008-02-07 |
AU2003300192A1 (en) | 2004-07-22 |
DE60323153D1 (en) | 2008-10-02 |
EP1583556A4 (en) | 2006-04-26 |
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