WO2012148024A1 - Amorce et sonde pour détecter la résistance au tamiflu dans une nouvelle souche de la grippe a/h1n1, et procédé de diagnostic utilisant celles-ci - Google Patents

Amorce et sonde pour détecter la résistance au tamiflu dans une nouvelle souche de la grippe a/h1n1, et procédé de diagnostic utilisant celles-ci Download PDF

Info

Publication number
WO2012148024A1
WO2012148024A1 PCT/KR2011/003240 KR2011003240W WO2012148024A1 WO 2012148024 A1 WO2012148024 A1 WO 2012148024A1 KR 2011003240 W KR2011003240 W KR 2011003240W WO 2012148024 A1 WO2012148024 A1 WO 2012148024A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
tamiflu
influenza
single nucleotide
nucleotide polymorphism
Prior art date
Application number
PCT/KR2011/003240
Other languages
English (en)
Korean (ko)
Inventor
김동식
변상진
이충현
김미란
김성열
박한오
Original Assignee
㈜바이오니아
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by ㈜바이오니아 filed Critical ㈜바이오니아
Publication of WO2012148024A1 publication Critical patent/WO2012148024A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a primer, a probe for detecting Tamiflu resistance of H1N1 influenza A, and a diagnostic method using the same.
  • Swine Influenza is a respiratory disease called swine flu or swine influenza.
  • the World Health Organization (WHO) has replaced swine flu (H1N1) as “Influenza A (H1N1)” in 2009. Named as of April 30.
  • This new swine influenza A virus was first discovered in April 2009, and can cause infection in humans, pigs, and birds.
  • the path of infection is mainly respiratory infections and contact infections through coughing or sneezing of infected patients. All respiratory secretions, body fluids (including diarrhea and feces) have been reported to be potentially infectious.
  • the virus breaks down sialic acid, which is external to the cell, using neuraminidase made by the virus in order to create a new virus in the cell after infection and spread to other cells. It is necessary.
  • Tamiflu-resistant avian influenza viruses are known to occur through three genetic variations (Arg292Lys, Asn294Ser, His274Tyr) that normally alter the amino acids of Tamiflu's receptor protein to which Tamiflu binds.
  • the 274th amino acid the most highly expressed variant of the Tamiflu-resistant avian influenza virus, is a variant in which Thoflu's hydrophobic residues do not bind to Tamiflu receptors. If it is. It is a virus found in the United States and Europe in the 10 to 25% probability of the 2007-2008 season, increasing the risk of future mutations for swine flu. The emergence of these Tamiflu-resistant viruses therefore provides a justification for humans to continue developing new viral therapies.
  • SNPs for detecting single nucleotide polymorphism which are widely used in molecular genetics, include sequencing, PCR-SSCP (Polymerase chain reaction-Single stranded conformation polymorphism), allele specific hybridization, and ollie. There are oligo-ligation, mini-sequencing and enzymatic cleavage. A method using a DNA chip has also been introduced, which is in principle no different from an allele specific hybridization, but differentiated from a stationary phase to which an oligonucleotide probe is attached.
  • the sequencing method includes the Macsam-Gilbert method and the Sanger method, but the latter method is mainly used.
  • This method is not only to investigate the genetic variation of a specific position, but to identify the nucleotide sequence of all or a part of the gene, but if the nucleotide sequence is revealed, it can be used for SNP scoring because it can also identify the genetic variation of a specific position. have.
  • it is inefficient in that it reads not only the mutation of the SNP to be investigated but also reads the nucleotide sequence of the surrounding which does not need to be investigated.
  • PCR-SSCP (Orita, M. et.al, Genomics , 1989, 5: 8874-8879) amplifies the sequences containing the SNPs to be analyzed by PCR, separates them into their respective chains, and then polyacrylamide gels. Perform electrophoresis at. Since the secondary structure of the DNA chain is changed by one base difference, the base variation is examined according to the electrophoretic migration speed caused by this difference.
  • Allele specific hybridization is a method of hybridizing sample DNA labeled with a radioisotope to a probe attached to a nylon filter or the like, and examining whether or not there is a base mutation by controlling conditions such as temperature and hybridization.
  • Real-Time Polymerase Chain Reaction is a type of polymerase chain reaction method that checks amplification according to the intensity of a fluorescence signal using a primer and a probe.
  • the applicant also has a Ct value obtained from a reaction product obtained by performing a real-time polymerase chain reaction using an alleleic specific primer and a probe, more specifically, a Ct value ⁇ ⁇ 2, heterozygotes, and Ct values> ⁇ 2, in Korean Patent No. 2011-0004724, which identifies alleles of the target gene with the co-zygote, “A single base polymorphism of the target gene is determined by PCR. Method ”.
  • the ASP (Allelic Specific Primer) method used in the present invention is a method of amplifying a site including a single base polymorphism using a polymerase chain reaction device and comparing the degree of amplification to determine a single base polymorphism.
  • the present inventors apply a method for determining a single nucleotide polymorphism of a target gene through PCR and a composition for a hot start PCR including blocking oligonucleotides.
  • the present invention aims to provide an efficient and accurate detection of a single nucleotide polymorphism site of a living organism having ribonucleic acid, which makes up for the shortcomings of the method for analyzing nucleotide polymorphism.
  • the present invention provides a single nucleotide polymorphism discriminative primer set capable of detecting a single nucleotide polymorphism of the Tamiflu-resistant C823T position of swine influenza A H1N1.
  • the present invention also provides a single nucleotide polymorphism discriminative probe capable of detecting a single nucleotide polymorphism at the Tamiflu-resistant C823T position of swine influenza A H1N1.
  • the present invention comprises a primer set for determining the single nucleotide polymorphism of the Tamiflu-resistant C823T position of the swine flu type A H1N1, a single nucleotide polymorphism probe of the Tamiflu-resistant C823T position of the H1N1 influenza A type H1N1, or a mixed construct thereof
  • a Tamiflu resistant C823T single nucleotide polymorphism discrimination kit of influenza A type H1N1 Provided are a Tamiflu resistant C823T single nucleotide polymorphism discrimination kit of influenza A type H1N1.
  • the present invention also provides a method for determining swine influenza resistant to Tamiflu by using the Tamiflu resistant C823T single nucleotide polymorphism discrimination kit of the swine influenza A type H1N1.
  • primer refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product.
  • the length and sequence of the primer should allow to start the synthesis of the extension product.
  • the specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.
  • each primer in order to detect C823T single nucleotide polymorphism (SNP), each primer is a specific polynucleotide or a complementary polynucleotide thereof which detects a sensitive and resistant virus, respectively, and has a length of 10 It may consist of from 30 nucleotides.
  • the polynucleotide is a polymorphic sequence.
  • a polymorphic sequence refers to a sequence comprising a polymorphic site representing a single nucleotide polymorphism in a nucleotide sequence.
  • a polymorphic site refers to a site where a single nucleotide polymorphism occurs in the polymorphic sequence.
  • the polynucleotide can be DNA or RNA.
  • the primer is 630 to the influenza virus A / Korea / 01/2009 (H1N1) (NCBI GenBank No: GQ132185) and A / Denmark / 528/2009 (H1N1) (NCBI GenBank No: CY043352) gene
  • H1N1 NCBI GenBank No: GQ132185
  • H1N1 NCBI GenBank No: CY043352
  • a polynucleotide consisting of a nucleotide within a 1027th base or a complementary polynucleotide thereof and more specifically, a primer set consisting of a polynucleotide consisting of 10 or more consecutive DNA sequences including the 823th base or a complementary nucleotide thereof. do.
  • the primer set includes a primer set of all combinations consisting of forward and reverse primers for recognizing a target gene sequence, but preferably, the primer set provides an analysis result having specificity and sensitivity. May be made by mixing two or more primers selected from the primers set forth in SEQ ID NOs: 1 to 6, 9, and 10.
  • the primer set is SEQ ID NO: 1, 2, 9 and 10; SEQ ID NO: 3, 4, 9 and 10; characterized in that at least one kind.
  • SEQ ID NO: 9 and 10 is a blocking oligonucleotide having a 3 'terminal hydroxyl group is blocked and has a nucleotide sequence complementary to the primer, more specifically, the blocking oligonucleotide is a hydroxyl group of 3' terminal is substituted with a substituent other than the hydroxyl group Substituted, preferably substituted with amines, complementary to susceptibility and resistant viral sequences, and consist of polynucleotides designed with a low Tm value of 5 to 10 ° C.
  • the primer set of the present invention includes a blocking oligonucleotide, thereby suppressing a primer band dimer formation at a low temperature or a smeared band of a PCR amplification product due to a nonspecific reaction, thereby obtaining a more accurate PCR result.
  • the blocking oligonucleotide may specifically bind to any one of a forward primer and a reverse primer with respect to a primer set having a sequence complementary thereto, or may specifically bind to both the forward and reverse primers.
  • the present invention provides C823T single nucleotide polymorphism (SNP) in influenza virus A / Korea / 01/2009 (H1N1) (NCBI GenBank No: GQ132185) and A / Denmark / 528/2009 (H1N1) (NCBI GenBank No: CY043352) gene.
  • SNP single nucleotide polymorphism
  • the probe is at least one selected from the probes set forth in SEQ ID NO: 7, SEQ ID NO: 8.
  • probe refers to nucleic acid fragments such as RNA or DNA corresponding to short bases to hundreds of bases that can achieve specific binding with mRNA, and are labeled to confirm the presence or absence of specific mRNAs.
  • Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like.
  • a fluorescent labeled probe can be used. More specifically, the fluorescent material is coupled to the 5 'end, and the quencher may be coupled to the 3' end.
  • the present invention includes a primer set for determining single nucleotide polymorphism at the Tamiflu-resistant C823T position of the swine flu type A H1N1, a single nucleotide polymorphism probe at the Tamiflu-resistant C823T position of the H1N1 influenza A type H1N1, or a mixed construct thereof.
  • a Tamiflu resistant C823T single nucleotide polymorphism discriminative kit of type H1N1 is provided.
  • the mixed construct is characterized in that SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10.
  • the kit may also include instructions.
  • the guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented.
  • the instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit.
  • the guide includes information that is disclosed or provided through electronic media such as the Internet.
  • the present invention is a.
  • step 2 2) comparing the presence or absence of the polymorphic site detected in step 1);
  • It provides a method for determining the influenza, including a Tamiflu resistant.
  • sample includes samples such as blood, serum, saliva, or sputum, in which the HA and / or influenza A gene expression levels of swine influenza A H1N1 differ due to influenza virus infection, It is not limited to this.
  • the mixed construct is characterized in that SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10.
  • a single nucleotide polymorphism discrimination test was performed on a swine flu A (HN1) positive sample using primers and probes according to the present invention.
  • Virus RNA was extracted with Accuprep Viral RNA Extration kit ( Bionia , Korea) and subjected to real-time reverse transcription polymerase chain reaction using Exicycler TM Quantitative Thermal Block.
  • Accuprep Viral RNA Extration kit Bionia , Korea
  • Exicycler TM Quantitative Thermal Block Exicycler TM Quantitative Thermal Block.
  • Tamiflu-resistant C823T single nucleotide polymorphism discriminating primer sets and probes of the H1N1 influenza A type according to the present invention can not only accurately and efficiently analyze genetic variation of living organisms with ribonucleic acid but also Tamiflu resistance of the H1N1 influenza A type H1N1.
  • the C823T single nucleotide polymorphism site can be efficiently and accurately detected in a short time, greatly contributing to the development of new viral therapeutics such as Tamiflu-resistant virus.
  • Figure 1 shows the nucleotide sequence of influenza virus A / Korea / 01/2009 (H1N1) (NCBI GenBank No: GQ132185) according to the present invention
  • Figure 2 shows the nucleotide sequence of influenza virus A / Denmark / 528/2009 (H1N1) (NCBI GenBank No: CY043352) according to the present invention
  • Figure 3 shows the results (graph and detection sensitivity) of the real-time reverse transcription polymerase chain reaction for the primer set (10p) described in SEQ ID NO: 1 and SEQ ID NO: 5 according to the present invention
  • Figure 4 shows the results of the real-time reverse transcription polymerase chain reaction for the primer set (15p) described in SEQ ID NO: 1 and SEQ ID NO: 5 according to the present invention (graph and detection sensitivity),
  • Figure 5 shows the results (graph and detection sensitivity) of the real-time reverse transcription polymerase chain reaction for the primer set (10p) described in SEQ ID NO: 1 and SEQ ID NO: 6 according to the present invention
  • Figure 7 shows the results (graph and detection sensitivity) of the real-time reverse transcription polymerase chain reaction for the primer set (10p) described in SEQ ID NO: 3 and SEQ ID NO: 5 according to the present invention
  • FIG. 10 shows the results (graph and detection sensitivity) of real-time reverse transcriptase polymerase chain reaction for primer sets (15p) described in SEQ ID NO: 3 and SEQ ID NO: 6 according to the present invention.
  • FIG. 11 shows the results (graph and detection sensitivity) of real-time reverse transcriptase polymerase chain reaction for primer sets (10p) described in SEQ ID NO: 2 and SEQ ID NO: 5 according to the present invention
  • FIG. 13 shows the results (graph and detection sensitivity) of real-time reverse transcriptase polymerase chain reaction for primer sets (10p) described in SEQ ID NO: 2 and SEQ ID NO: 6 according to the present invention.
  • 21 shows the results (graph and detection sensitivity) of real-time reverse transcriptase polymerase chain reaction of the primer set of SEQ ID NO: 4 and SEQ ID NO: 6 and the probe of SEQ ID NO: 7 according to the present invention
  • FIG. 23 is a graph of a single nucleotide polymorphism sensitive RNA standard template of Swine Flu for Example 3 of the present invention using Exicycler TM 96 Real-Time Quantitative Thermal Block,
  • FIG. 24 shows a graph of the standard template for single nucleotide polymorphism resistance of swine flu against Example 3 of the present invention using Exicycler TM 96 Real-Time Quantitative Thermal Block,
  • FIG. 25 shows standard curves of a standard template graph of susceptibility / tolerance of single nucleotide polymorphism of Swine Flu for Example 3 of the present invention using Exicycler TM 96 Real-Time Quantitative Thermal Block
  • FIG. 26 shows the detection sensitivity ( ⁇ Ct) of real-time reverse transcription polymerase chain reaction of the susceptible standard template 10 5 of FIG. 23 using Exicycler TM 96 Real-Time Quantitative Thermal block.
  • FIG. 27 shows the detection sensitivity ( ⁇ Ct) of real-time reverse transcription polymerase chain reaction for the resistant standard template 10 5 of FIG. 24 using Exicycler TM 96 Real-Time Quantitative Thermal block.
  • FIG. 28 shows the results of detection of resistant single nucleotide polymorphism of a new influenza A (HN1) negative sample for Example 4 of the present invention using an Exicycler TM 96 Real-Time Quantitative Thermal block.
  • FIG. 29 shows the results of detection of resistant single nucleotide polymorphism of Swine Influenza A (HN1) positive samples for Example 4 of the present invention using Exicycler TM 96 Real-Time Quantitative Thermal Block.
  • H1N1 susceptible influenza A
  • H1N1 susceptible influenza A
  • NCBI GenBank No: GQ132185 SEQ ID NO: 11
  • a / Denmark / 528/2009 H1N1
  • resistant to influenza A H1N1
  • NCBI GenBank No: CY043352 SEQ ID NO: 12
  • the reaction solution was inoculated into LB plates containing ampicillin, IPTG, and X-Gal, followed by incubation at 37 ° C for 16 hours.
  • the cultured white colonies were taken, cultured in LB liquid medium for 16 hours, centrifuged, the supernatant was discarded, and plasmid DNA was extracted from the pellets using Accuprep plasmid DNA prep kit ( Bionia ).
  • the plasmid DNA was measured using a UV spectrometer (manufactured by Shimazu, Japan) to measure the concentration and purity, and confirmed that the purity was between 1.8 and 2.0.
  • the plasmid DNA was transcribed into RNA using the MAXIscipt In vitro Transcription Kit (Ambion, USA). .
  • RNA copy number was calculated by the following formula.
  • H1N1 A / Korea / 01/2009 (H1N1), the susceptible influenza A (H1N1) of SEQ ID NO: 11 (NCBI GenBank No: GQ132185, and A / Denmark / 528/2009 (H1N1), the resistant influenza A (H1N1) of SEQ ID NO: 12
  • primers for cDNA synthesis were randomly selected such that the primers had a length of 24 to 25mer and a Tm value of 55 ° C to 60 ° C as reverse primers (SEQ ID NO: 5, SEQ ID NO: 6)
  • the blocking oligonucleotide to prevent the action of a single nucleotide polymorphism in the cDNA synthesis step was designed to be 17mer, Tm value is 45 °C, the cDNA synthesis Tm (SEQ ID NO: 9, SEQ ID NO: 10).
  • Specific susceptible / resistant forward primers (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) for detecting single nucleotide polymorphisms using cDNA between 23-24mer in length and 55-60 ° C in Tm
  • the forward nucleotide sequence was selected so that the single base polymorphic portion was located at the 3 ′ end, and the probe (SEQ ID NO: 7, SEQ ID NO: 8) length was a Tm 60 to 65 ° C. probe between 24 and 26mer. Tm values were checked using the Primer3Plus program.
  • the primers of Table 1 were combined into a set of Table 2 below to perform real-time reverse transcription polymerase chain reaction using Exicycler TM Quantitative Thermal Block (Bionia, Korea).
  • the blocking oligonucleotide AccPower GreenStar TM qPCR Premix the template RNA used in DW 45 ⁇ l here using (( ⁇ ) Bioneer Korea) (scale of 10 5) was diluted in the above Example 1
  • the final total dose was 50 ⁇ l and the experiment was performed in the combination as shown in Table 2 above.
  • primer set 4 could be selected from the results of FIGS. 3 to 18.
  • Probe with good PCR efficiency was selected using the selected primer set 4.
  • the probe was used by custom fabrication (Bionia Co., Ltd.) to attach FHQ fluorescence at the 5 ′ region and BHQ1 at the 3 ′ region.
  • the probes were prepared in the combinations shown in Table 3 below, and 2 ⁇ l of 10X RT Buffer, Taq polymerase 1U, MMLV RTase 350 U (above Bionic), dNTP 20 mM, DTT 50 mM, RNasin 15U, and DEPC (Diethylpyrocarbonate) DW, respectively. After mixing and mixing, the mixture was made to 45 ⁇ l per well, and the resultant was dispensed into a 96-well plate and reacted at 45 ° C. for 15 minutes to synthesize cDNA. 45 cycles were reacted for 5 seconds.
  • RNA prepared in Example 1 was used as a template, and real-time reverse transcription polymerase chain reaction was performed using the primers and probes selected in Example 2 and Exicycler TM Quantitative Thermal Block (Bionia, Korea).
  • the number of copies was calculated according to the method of Example 1, as a result template RNA was able to detect a minimum of 10 copies, to prepare a standard graph of the standard template real-time reverse transcription polymerase chain reaction
  • the slope was -3.01 to -3.15, and the linearity R 2 value was 0.997 to 0.999.
  • the R 2 is a correlation coefficient indicating the linearity of the graph when the standard graph of the real-time polymerase chain reaction is drawn, and the closer to 1 (the closer to the straight line), the PCR was properly performed.
  • Example 2 Using the primers and probes prepared in Example 2, a single nucleotide polymorphism discrimination test was performed on Swine Flu A (HN1) positive specimens (provided by Chungnam National University Hospital). Virus RNA was extracted with an Accuprep Viral RNA Extration kit ( Bionia , Korea) and subjected to real-time reverse transcription polymerase chain reaction under the same conditions as in Example 2 using Exicycler TM Quantitative Thermal Block. This is to confirm whether the same result is obtained when the primer and probe of the present invention are applied to the actual sample as well as the standard template of Example 3.
  • influenza A virus Inf A
  • influenza B virus Inf B
  • avian influenza virus AIV
  • HCV hepatitis C virus
  • Noro RNA
  • EV virus EV virus
  • HTLV 1 virus HTLV 1 virus
  • the Tamiflu-resistant C823T single nucleotide polymorphism discriminating primer set and probe of the H1N1 influenza A type H1N1 can accurately and efficiently analyze the genetic variation of a living organism having ribonucleic acid, as well as the H1N1 influenza A type.
  • the Tamiflu-resistant C823T single nucleotide polymorphism site of H1N1 can be efficiently and accurately detected in a short time, greatly contributing to the development of new viral therapeutics such as Tamiflu-resistant virus.
  • SEQ ID NO: 1 is a nucleotide sequence of primer SIV_M_wild forward primer 1 for detecting single nucleotide polymorphism of the Tamiflu resistant C823T position of H1N1 influenza A according to the present invention.
  • SEQ ID NO: 2 is a nucleotide sequence of primer SIV_M_mutant forward primer 1 for detecting single nucleotide polymorphism of the Tamiflu resistant C823T position of H1N1 influenza A according to the present invention.
  • SEQ ID NO: 3 is a nucleotide sequence of primer SIV_M_wild forward primer 2 for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the new influenza A type H1N1 according to the present invention.
  • SEQ ID NO: 4 is a nucleotide sequence of primer SIV_M_mutant forward primer 2 for detecting single nucleotide polymorphism of the Tamiflu resistant C823T position of H1N1 influenza A according to the present invention.
  • SEQ ID NO: 5 is a nucleotide sequence of a primer SIV reverse primer 1 for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the H1N1 influenza A according to the present invention.
  • SEQ ID NO: 6 is a nucleotide sequence of primer SIV reverse primer 2 for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the new influenza A type H1N1 according to the present invention.
  • SEQ ID NO: 7 is a nucleotide sequence of the probe SIV probe 1 for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the new influenza A type H1N1 according to the present invention.
  • SEQ ID NO: 8 is a nucleotide sequence of the probe SIV probe 2 for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the new influenza A type H1N1 according to the present invention.
  • SEQ ID NO: 9 is a nucleotide sequence of a primer wild blocking primer for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the new influenza A type H1N1 according to the present invention.
  • SEQ ID NO: 10 is a nucleotide sequence of a primer mutant blocking primer for detecting a single nucleotide polymorphism of the Tamiflu resistant C823T position of the new influenza A type H1N1 according to the present invention.
  • SEQ ID NO: 11 is a nucleotide sequence of influenza virus A / Korea / 01/2009 (H1N1) (NCBI GenBank No: GQ132185) according to the present invention.
  • SEQ ID NO: 12 is a nucleotide sequence of influenza virus A / Denmark / 528/2009 (H1N1) (NCBI GenBank No: CY043352) according to the present invention.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une amorce et une sonde pour détecter la résistance au Tamiflu dans une nouvelle souche de la grippe A/H1N1, et un procédé de diagnostic utilisant celles-ci. Un ensemble d'amorce et une sonde permettant la discrimination du polymorphisme simple nucléotide C823T résistant au Tamiflu pour une nouvelle souche de la grippe A/H1N1 selon la présente invention constituent une contribution importante au développement de nouveaux médicaments antiviraux pour des virus résistants au Tamiflu et similaire, étant donné que non seulement ils permettent l'analyse précise et efficace de la variation génétique dans des formes de vie à acide ribonucléique mais ils permettent également de détecter efficacement et précisément le site du polymorphisme simple nucléotide C823T résistant au Tamiflu dans une nouvelle souche de la grippe A/H1N1 dans un temps court.
PCT/KR2011/003240 2011-04-29 2011-04-29 Amorce et sonde pour détecter la résistance au tamiflu dans une nouvelle souche de la grippe a/h1n1, et procédé de diagnostic utilisant celles-ci WO2012148024A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2011-0040494 2011-04-29
KR20110040494A KR101329810B1 (ko) 2011-04-29 2011-04-29 신종 인플루엔자 ａ형 ｈ１ｎ１의 타미플루 내성 검출용 프라이머, 프로브 및 이를 이용한 진단 방법

Publications (1)

Publication Number Publication Date
WO2012148024A1 true WO2012148024A1 (fr) 2012-11-01

Family

ID=47072527

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2011/003240 WO2012148024A1 (fr) 2011-04-29 2011-04-29 Amorce et sonde pour détecter la résistance au tamiflu dans une nouvelle souche de la grippe a/h1n1, et procédé de diagnostic utilisant celles-ci

Country Status (2)

Country Link
KR (1) KR101329810B1 (fr)
WO (1) WO2012148024A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101575747B1 (ko) * 2015-04-06 2015-12-21 한국생명공학연구원 항바이러스제-감수성/저항성 바이러스 검출 시스템
WO2017007204A1 (fr) * 2015-07-03 2017-01-12 한국생명공학연구원 Système de détection de virus résistant à un agent antiviral
KR101788454B1 (ko) 2015-07-03 2017-10-19 한국생명공학연구원 항바이러스제-저항성 바이러스 검출 시스템
KR101969426B1 (ko) 2017-05-30 2019-04-16 주식회사 퀀타매트릭스 Sti 원인균 또는 바이러스 검출용 조성물

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070087338A1 (en) * 2005-10-17 2007-04-19 Rangarajan Sampath Compositions for use in identification of influenza viruses
US7695941B2 (en) * 2005-06-16 2010-04-13 The United States Of America As Represented By The Secretary Of The Navy Multiplexed polymerase chain reaction for genetic sequence analysis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7695941B2 (en) * 2005-06-16 2010-04-13 The United States Of America As Represented By The Secretary Of The Navy Multiplexed polymerase chain reaction for genetic sequence analysis
US20070087338A1 (en) * 2005-10-17 2007-04-19 Rangarajan Sampath Compositions for use in identification of influenza viruses

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHIDLOW, G. R. ET AL.: "The detection of oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using a real-time RT-PCR assay", JOURNAL OF VIROLOGICAL METHODS, vol. 169, 30 June 2010 (2010-06-30) *
LEE, YO HAN ET AL.: "A Case of Oseltamivir-Resistant Pandemic Influenza (H1N1 2009) in a Patient with Acute Myeloid Leukemia", INFECT. CHEMOTHER., vol. 42, no. 2, April 2010 (2010-04-01) *
VRIES, E. ET AL.: "Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase", JOURNAL OF CLINICAL VIROLOGY, vol. 47, 25 October 2009 (2009-10-25) *

Also Published As

Publication number Publication date
KR101329810B1 (ko) 2013-11-15
KR20120122385A (ko) 2012-11-07

Similar Documents

Publication Publication Date Title
WO2018174318A1 (fr) Procédé d'analyse de courbes de fusion à l'aide d'une sonde pna bifonctionnelle, et procédé de diagnostic de l'instabilité des microsatellites et kit de diagnostic de l'instabilité des microsatellites utilisant ledit procédé de diagnostic
KR20120095256A (ko) 호흡기 바이러스 검출용 조성물 및 이를 포함하는 호흡기 바이러스 검출용 키트
KR102338861B1 (ko) RT-LAMP를 이용한 코로나바이러스감염증-19을 일으키는 SARS-CoV-2의 검출용 PNA 프로브와 프라이머 및 이를 이용한 감염여부 판별방법
WO2017122897A1 (fr) Marqueur génétique servant à détecter le virus responsable de l'iridovirose de la daurade japonaise, et procédé de détection du virus causal utilisant le marqueur
Shahrajabian et al. Different methods for molecular and rapid detection of human novel coronavirus
WO2012148024A1 (fr) Amorce et sonde pour détecter la résistance au tamiflu dans une nouvelle souche de la grippe a/h1n1, et procédé de diagnostic utilisant celles-ci
CN110387439B (zh) 用于腺病毒检测与分型的引物和探针、试剂盒及方法
WO2021177773A2 (fr) Composition pour le diagnostic du sars-cov-2, kit, et procédé pour le diagnostic du sars-cov-2 l'utilisant
WO2022014867A1 (fr) Réaction en chaîne par polymérase de transcription inverse en temps réel à regulation positive pour empêcher l'apparition de faux positifs
WO2022005255A2 (fr) Kit et méthode de diagnostic simultané du sars-cov-2 induisant la maladie à coronavirus-19 et du sarbecovirus faisant appel à une sonde pna
CN116694743B (zh) 一种利用荧光探针检测多靶标基因序列的方法
CN111100924B (zh) 一种用于检测fmr1基因cgg重复数的质控品及其应用和含有该质控品的试剂盒
WO2020060170A1 (fr) Composition de marqueur pour le diagnostic de dermatite atopique et procédé, utilisant celle-ci, pour la prédiction ou le diagnostic de dermatite atopique
KR20230062511A (ko) 코로나바이러스-19 검출용 조성물 및 이의 검출용 키트
WO2010137873A2 (fr) Amorce pour le diagnostic du nouveau virus de la grippe a, sonde, kit la comprenant et méthode de diagnostic faisant appel audit kit
WO2011142646A9 (fr) Procédé de détection de hpv (papillomavirus humain) et de son génotype
CN111235268B (zh) Snp位点基因型检测试剂及相应的试剂盒中的用途及试剂盒
WO2012002594A1 (fr) Amorce destinée à diagnostiquer le virus de l'hépatite c, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite c utilisant le kit
WO2021215556A1 (fr) Procede de preparation d'un kit de diagnostic de coronavirus, kit de diagnostic de coronavirus ainsi preparé, et procédé de diagnostic de coronavirus l'utilisant
EP2245185A1 (fr) Une mutation dans la région régulatrice du gène gjb2 responsable de surdité néonatale au sein de dfnb1
CN114807432B (zh) 快速检测新型冠状病毒及其Delta突变株的试剂盒和方法
CN112126713A (zh) 一种冠状病毒和流感病毒联合检测产品及其用途
KR20100096807A (ko) 아토피 피부염 진단용 마커 및 그의 용도
WO2008134933A1 (fr) Trousse de fluorescence à deux couleurs utiles pour détecter la réaction en chaîne de la polymérase de coamplification
WO2012070788A9 (fr) Procédé et trousse pour la quantification d'acides nucléiques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11864422

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11864422

Country of ref document: EP

Kind code of ref document: A1