[go: up one dir, main page]

WO2013183572A1 - Procédé de mesure de l'activité inhibitrice d'un inhibiteur de polymérase - Google Patents

Procédé de mesure de l'activité inhibitrice d'un inhibiteur de polymérase Download PDF

Info

Publication number
WO2013183572A1
WO2013183572A1 PCT/JP2013/065299 JP2013065299W WO2013183572A1 WO 2013183572 A1 WO2013183572 A1 WO 2013183572A1 JP 2013065299 W JP2013065299 W JP 2013065299W WO 2013183572 A1 WO2013183572 A1 WO 2013183572A1
Authority
WO
WIPO (PCT)
Prior art keywords
reverse transcriptase
labeling substance
template
primer
polymerase
Prior art date
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.)
Ceased
Application number
PCT/JP2013/065299
Other languages
English (en)
Japanese (ja)
Inventor
了 森下
澤崎　達也
遠藤　弥重太
明秀 梁
山本　直樹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CellFree Sciences Co Ltd
Original Assignee
CellFree Sciences Co Ltd
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 CellFree Sciences Co Ltd filed Critical CellFree Sciences Co Ltd
Publication of WO2013183572A1 publication Critical patent/WO2013183572A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C12Q1/68—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
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • the present invention relates to a method for measuring the inhibitory activity of a polymerase inhibitor, particularly a reverse transcriptase inhibitor. More specifically, by using a primer containing a labeling substance, and a template containing the primer binding site sequence, inhibitor complementary sequence, and polymerase activity detection sensitivity enhancement site sequence, simple, rapid and reliable inhibition of the polymerase inhibitor is performed.
  • the present invention relates to a method for measuring activity. This application claims priority from Japanese Patent Application No. 2012-126648, which is incorporated herein by reference.
  • HIV Human immunodeficiency virus
  • Retroviruses such as HIV synthesize complementary DNA using their RNA gene as a template as the first step of proliferation after infection of cells.
  • the enzyme that works in this case is reverse transcriptase.
  • the establishment of HAART therapy as a treatment for HIV has made it possible to delay the onset of AIDS.
  • the therapeutic effect is limited due to the emergence of resistant viruses and side effects. More specifically, due to the high rate of mutation expression, which is a characteristic of viruses, particularly the rate of mutation introduction into reverse transcriptase, resistant viruses to the administered drug easily appear. It has also been reported that viruses resistant to multiple drugs based on the introductory appearance have caused difficulties in the subsequent treatment.
  • Non-patent Document 1 Genotyping and Phenotyping drug resistance test methods have been established as effective anti-HIV-1 drug selection methods in HIV treatment.
  • Genotyping performs base sequence analysis by amplification of the HIV-1 pol gene region. However, it has often been reported that the prediction of drug resistance due to amino acid mutations in the gene encoding the reverse transcriptase or protease of the viral genome in the sample does not match the pharmacological drug resistance results of the patient.
  • Phenotyping is a method for directly testing drug resistance.
  • a virus drug resistance test using cells may take several months to obtain a result, which requires a lot of labor and a large amount of money, and is not suitable for processing a large number of specimens.
  • Patent Document 1 JP-A-2002-191399 (Patent Document 1) states that “an animal cell capable of expressing a secreted reporter protein by HIV-1 infection is brought into contact with a sample containing HIV-1 in the presence of a test drug. Discloses a method for testing HIV drug resistance, which comprises detecting a reporter protein secreted into the culture supernatant by infection. However, the method for detecting HIV drug resistance described in this publication is clearly different from the method of the present invention because it uses HIV-1-infected cells.
  • Patent Document 2 JP 2002-508158 discloses "a detection method of HIV drug resistance characterized by identifying a colony containing a drug resistance target protein based on a reporter mechanism of a reporter protein".
  • the method for detecting HIV drug resistance described in this publication is clearly different from the method of the present invention because it uses a bacterial reporter system.
  • Patent Document 3 discloses a method for selecting an optimal antiviral agent.
  • the reverse transcriptase primers and templates described in this publication are clearly different from the primer and template structures used in the present invention.
  • kits are commercially available as methods for measuring reverse transcriptase activity derived from retroviruses such as HIV. Quan-T-RT assay system (GE Healthcare), EnzChek® Reverse Transcriptase Assay Kit (Invitrogen), Lenti RT Activity Kit (Cavidi AB), Reverse Transcriptase Assay, Colorimetric (Roche Applied Science)
  • the template is poly-A, and the structure is clearly different from the template of the present invention.
  • the kit is intended to measure reverse transcriptase activity.
  • Any kit can measure the inhibitory activity of non-nucleic acid or thymidine analog nucleic acid reverse transcriptase inhibitors, but cannot measure the inhibitory activity of other nucleic acid analogs. It is necessary to prepare a polynucleotide of a nucleic acid analog showing complementarity as a template.
  • an object of the present invention is to provide a method for measuring the inhibitory activity of a simple, rapid and reliable polymerase inhibitor, particularly a reverse transcriptase inhibitor.
  • the present inventors have used a primer containing a labeling substance and a template containing the primer binding site sequence, inhibitor complementary sequence, and polymerase activity detection sensitivity enhancing site sequence to obtain a polymerase inhibitor.
  • the present inventors completed the present invention by discovering that drug resistance can be detected easily, quickly and with high reliability. That is, the present invention provides a method for selecting a tailor-made polymerase inhibitor that is optimal for individual patients.
  • a method for measuring the inhibitory activity of a polymerase inhibitor comprising: Contacting a polymerase, one or more polymerase inhibitors, a template, a primer containing a labeling substance, and a nucleotide containing a labeling substance, and measuring the polymerase activity of the polymerase;
  • the template contains YSD
  • Y is a primer binding site sequence
  • S is an inhibitor complementary sequence
  • D is a polymerase activity detection sensitivity enhancement site sequence of the polymerase
  • the primer contains a labeling substance.
  • a measurement method wherein X is X, Y is hybridized to Y, and X and Y are any of DNA, RNA, and chimeric polynucleotides.
  • the template contains 3′-YSD-5 ′, and the primer contains X-3 ′ containing a 5′-labeling substance.
  • the polymerase is a reverse transcriptase. 4).
  • the reverse transcriptase activity of the reverse transcriptase is measured by ALPHA (Amplified Luminescence Proximity Homogeneous Assay). 5.
  • the measurement method includes the following steps: (1) Recognizing directly or indirectly a reverse transcriptase, one or a plurality of reverse transcriptase inhibitors, a template, a primer containing a labeling substance, a nucleotide containing a labeling substance, a labeling substance of the primer or a labeling substance of the nucleotide Contacting a possible acceptor bead and a donor bead capable of directly or indirectly recognizing a labeling substance of the primer or a labeling substance of the nucleotide; (2) A step of detecting the activity of the reverse transcriptase by a change in signal intensity between the acceptor beads and the donor beads. 6).
  • step (1) is as follows: Reverse transcriptase, one or more reverse transcriptase inhibitors, the template, a primer containing DIG as a labeling substance, a nucleotide containing biotin as a labeling substance, acceptor beads capable of directly or indirectly recognizing the DIG, and Contacting a donor bead bound with avidin or streptavidin capable of directly or indirectly recognizing the biotin; Or reverse transcriptase, one or a plurality of reverse transcriptase inhibitors, the template, a primer containing biotin as a labeling substance, a nucleotide containing DIG as a labeling substance, and acceptor beads capable of directly or indirectly recognizing the DIG And a step of contacting donor beads to which avidin or streptavidin capable of directly or indirectly recognizing the biotin is bound.
  • a template for use in measuring the inhibitory activity of a reverse transcriptase inhibitor The template is 3′-YSD-5 ′, Y is a sequence for hybridizing with a primer, S is a sequence complementary to the inhibitor of the reverse transcriptase, and at least one of adenine, guanine, cytosine, and uracil A polynucleotide comprising any combination including the above, wherein D is a reverse transcriptase activity detection sensitivity enhancing site sequence of the reverse transcriptase, wherein D contains polyadenine. 10.
  • a kit for measuring the inhibitory activity of a reverse transcriptase inhibitor 10.
  • the present invention provides a simple, rapid and reliable method for measuring the inhibitory activity of an inhibitor when selecting a tailor-made polymerase inhibitor, particularly a reverse transcriptase inhibitor, which is optimal for an individual patient.
  • FIG. 1 Schematic diagram of primer and template of the present invention
  • Schematic diagram of detection of drug resistance of reverse transcriptase in vitro using ALPHA Measurement of reverse transcriptase activity in a measurement system using a primer containing a labeling substance Changes in reverse transcriptase activity depending on template / primer concentration
  • Inhibitory effect of reverse transcriptase activity by nucleic acid inhibitors or non-nucleic acid inhibitors Detection result of inhibitory activity of reverse transcriptase activity due to template difference Stanford Drug Resistance Database Confirmation of inhibitory activity against reverse transcriptase activity of drug-resistant reverse transcriptase by 3TCTP Confirmation of inhibitory activity of reverse transcriptase inhibitors against the reverse transcriptase activity of drug-resistant reverse transcriptase Improvement of detection sensitivity for inhibition of reverse transcriptase activity by adding ATP
  • the method for measuring the inhibitory activity of the polymerase inhibitor of the present invention mainly has the following characteristics.
  • (1) By using a primer containing a labeling substance and a template containing the primer binding site sequence, inhibitor complementary sequence, and polymerase activity detection sensitivity enhancing site sequence, the signal based on the polymerase activity is increased and the polymerase inhibitor is inhibited.
  • the detection sensitivity of activity increases. That is, by introducing the polymerase activity detection sensitivity enhancement site sequence into the template, not only the signal value but also the ratio of the signal value / noise value can be increased.
  • a transcription template is synthesized without introducing a polymerase into a vector, and is further expressed as a translation template by a cell-free protein synthesis system.
  • a polymerase can be expressed simply and rapidly.
  • the inhibitory activity of the polymerase inhibitor is preferably detected by a homogeneous assay. Thereby, since purification is not required in all steps, the inhibitory activity of the polymerase inhibitor can be measured simply, quickly and easily.
  • a non-nucleic acid system Regardless of the type of nucleic acid inhibitor, including inhibitors, the inhibitory activity of reverse transcriptase inhibitors can be measured with one type of template.
  • the measurement method of the present invention is characterized by using a primer including at least a labeling substance, and a template including the primer binding site sequence, an inhibitor complementary sequence, and a polymerase activity detection sensitivity enhancement site sequence (see FIG. 1).
  • nucleotide is a high molecular weight substance having as a structural unit a nucleotide which is a compound in which a phosphate group is ester-bonded to a sugar molecule of a nucleoside, which is a compound in which a base and a sugar are covalently bonded.
  • DNA is a polynucleotide composed of nucleotides having a sugar moiety that is deoxyribose and containing a base selected from the group consisting of adenine, guanine, cytosine, and thymine.
  • RNA is a polynucleotide composed of nucleotides whose sugar moiety is ribose and comprising a base selected from the group consisting of adenine, guanine, cytosine, and uracil.
  • Chimeric polynucleotide means a 2mer or more polynucleotide composed of nucleotides constituting DNA and nucleotides constituting RNA.
  • the expression “polynucleotide” simply means DNA, RNA, or chimeric polynucleotide.
  • the nucleotide sequence of the “primer containing a labeling substance (primer comprising X-3 ′ containing a 5′-labeling substance)” of the present invention is particularly limited as long as it can hybridize with the primer binding site sequence of the template described below. Not. Further, “including a labeling substance” means that a labeling substance is bonded directly or indirectly to any position of the nucleotide sequence of the primer, preferably to the 5 ′ end of the sequence.
  • the primer length is 5 to 40 mer, preferably 10 to 30 mer, more preferably 13 to 20 mer.
  • the template of the present invention includes at least a primer binding site sequence (Y), an inhibitor complementary sequence (S), and a polymerase activity detection sensitivity enhancement site sequence (D).
  • the nucleotide sequence of the primer binding site sequence (Y) is not particularly limited as long as it can hybridize with the primer containing the labeling substance described above.
  • the nucleotide sequence of the inhibitor complementary sequence (S) is the template sequence of the polymerase.
  • the polymerase is a DNA-dependent DNA polymerase, the sequence is an arbitrarily combined DNA sequence containing at least one of adenine, guanine, cytosine, and thymine.
  • the sequence is an arbitrarily combined RNA sequence containing at least one of adenine, guanine, cytosine, and uracil.
  • the sequence is an arbitrarily combined DNA sequence containing at least one of adenine, guanine, cytosine, and thymine.
  • the sequence is an RNA sequence arbitrarily combined containing at least one of adenine, guanine, cytosine, and uracil.
  • the nucleotide sequence length of the reverse transcriptase inhibitor complementary sequence (S) is 4 to 40 mer, preferably 8 to 20 mer.
  • the nucleotide sequence includes four types of nucleotides equally and arbitrarily combined.
  • the sequence is 12mer and RNA, it is a single-stranded RNA consisting of a sequence in which three adenines, three guanines, three cytosines, and three uracils are arbitrarily combined.
  • the nucleotide sequence of the polymerase activity detection sensitivity enhancement site sequence (D) is a nucleotide sequence for improving the probability that the polymerase incorporates the nucleotide containing the labeling substance (utilizes it for the polymerase activity).
  • the nucleotide of the nucleotide sequence of the polymerase activity detection sensitivity enhancing site sequence (D) is complementary to the nucleotide containing the labeling substance.
  • the adenine ratio of the polymerase activity detection sensitivity enhancing site sequence (D) is 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 99% or more. More preferably, the nucleotide sequence of the polymerase activity detection sensitivity enhancing site sequence (D) is composed of only adenine ⁇ poly (A) ⁇ .
  • the nucleotide sequence of the polymerase activity detection sensitivity enhancing site sequence (D) is preferably poly (A).
  • the nucleotide sequence length of the polymerase activity detection sensitivity enhancement site sequence (D) is 5 to 40 mer, preferably 8 to 30 mer, more preferably 10 to 20 mer.
  • the template of the present invention includes a primer binding site sequence (Y), an inhibitor complementary sequence (S), and a polymerase activity detection sensitivity enhancement site sequence (D), as well as other sequences or adducts (for immobilization on plates). May be added.
  • a preferred template used in the method for measuring the inhibitory activity of the reverse transcriptase inhibitor of the present invention is 3′-YSD-5 ′, and more specific sequences are RNA3 (AAAAAAUGCAGUCAGCUAGCUACGUGAACUCCAAGAUCCA: SEQ ID NO: 1), RNA4 (AAAAAAAAAAAAAAGCUAGCUACGUGAACUCCAAGAUCCA : SEQ ID NO: 2) and RNA5 (AAAAAAAAAAAAAAAGCUGCUCGUCGUGAACUCCAAGAUCCA: SEQ ID NO: 4), and a particularly preferred template is RNA5.
  • the nucleotide containing the labeling substance of the present invention means that the labeling substance is bound directly or indirectly to the nucleotide.
  • the nucleotide type is complementary to the nucleotide type of the nucleotide sequence of the polymerase activity detection sensitivity enhancing site sequence (D).
  • D polymerase activity detection sensitivity enhancing site sequence
  • Biotin-16-dUTP Biotin-16-2′-deoxy-uridine-5′-triphosphate
  • Biotin-20-dUTP Biotin-20-2′-deoxyuridine-5′-triphosphate
  • More preferably Biotin-16-dUTP More preferably Biotin-16-dUTP.
  • the “labeling substance” of the present invention is a substance that is directly or indirectly bound to a primer, nucleotide or the like in order to detect and measure the polymerase activity of the polymerase.
  • the following combinations are preferably used, but are not particularly limited as long as they can be used for detection of polymerase activity.
  • Examples of combinations of the labeling substance of the primer containing the labeling substance / the labeling substance of the nucleotide containing the labeling substance include DIG (Digoxigenin) / biotin, biotin / DIG (Digoxigenin), Dabcyl / FITC, and FITC / Dabcyl.
  • the “polymerase inhibitor” of the present invention means an agent that inhibits the polymerase activity of a virus-derived polymerase in a patient.
  • a polymerase inhibitor means a DNA polymerase inhibitor (particularly a reverse transcriptase inhibitor), an RNA polymerase inhibitor, or the like.
  • the “reverse transcriptase inhibitor” of the present invention means an agent that inhibits the reverse transcriptase activity of a reverse transcriptase derived from a virus (retrovirus, more specifically, a lentivirus) in a patient.
  • a virus retrovirus, more specifically, a lentivirus
  • therapeutic agents for HIV-1 infection, SIV infection, FIV infection, Visna virus infection, HBV infection, HSV infection and the like can be mentioned.
  • the “reverse transcriptase inhibitor” of the present invention also covers a reverse transcriptase inhibitor in the research stage or clinical trial stage that has not been put on the market at this stage.
  • nucleic acid reverse transcriptase inhibitors become active triphosphates, and are incorporated into viral DNA instead of dTTP, dATP, dCTP, dGTP, etc., respectively.
  • DNA chain elongation is stopped and virus growth is inhibited (see Table 1 below (types of nucleic acid reverse transcriptase inhibitors and non-nucleic acid reverse transcriptase inhibitors)).
  • Table 1 types of nucleic acid reverse transcriptase inhibitors and non-nucleic acid reverse transcriptase inhibitors
  • different templates single-stranded polynucleotides recognized by reverse transcriptase
  • the inhibitory activity of the reverse transcriptase inhibitor can be measured regardless of the type of the nucleic acid reverse transcriptase inhibitor and the non-nucleic acid reverse transcriptase inhibitor.
  • phosphorylation of the inhibitor that normally occurs in cells does not occur.
  • Type For example, TDF phosphate addition type TDFDP, 3TC phosphate addition type 3TCTP, and AZT phosphate addition type AZTTP are used.
  • the reverse transcriptase is preferably a virus-derived reverse transcriptase.
  • Viruses having reverse transcriptase include viruses belonging to the family Retroviridae. Lentivirus subfamily, oncovirus subfamily, spumavirus subfamily and the like are known as retroviridae.
  • the “reverse transcriptase” of the present invention includes a reverse transcriptase gene obtained from a patient sample, information on a known reverse transcriptase gene sequence, particularly information on a reverse transcriptase gene sequence known for drug resistance, a wild type reverse transcriptase gene Based on the sequence information, it can be expressed using a known protein expression system (in particular, a wheat germ cell-free protein synthesis system).
  • HIV-1 reverse transcriptase databases with known drug resistance include The French ANRS (National Agency for AIDS Research, http://www.hivfrenchresistance.org/index.html) and the Stanford University HIV Drug Resistance Database ( http://hivdb.stanford.edu/index.html). Furthermore, as the “reverse transcriptase” of the present invention, a reverse transcriptase obtained and purified from a patient sample can also be used.
  • sample means a biological material obtained directly from a human (or mammal) infected with each virus or after culturing.
  • Biological materials include, for example, all types of sputum, bronchial lavage, blood (plasma), skin tissue, biopsy, semen, lymphocyte blood culture, colony, liquid culture, fecal sample, urine, etc. May be. Particularly preferred is blood (plasma).
  • the patient sample means a fraction containing virus RNA extracted from the patient-derived sample (either in solution or in solid state).
  • Contact in each step of the present invention means that the A solution is added to the B solution or the B solution is added to the A solution.
  • the gene amplification solution may be added to the patient-derived sample, or the patient-derived sample may be added to the gene amplification solution.
  • a primer for amplifying a polymerase means a polynucleotide complementary to the polynucleotide strand to be amplified.
  • the primer is about 3 to 100 mer, preferably 5 to 70 mer, more preferably 10 to 50 mer.
  • the primer for amplifying the gene encoding polymerase is characterized by specifically hybridizing to a sequence other than the sequence portion involved in the physiological activity of the polymerase gene. Thereby, it is possible to detect mutations of all amino acids involved in the physiological activity of the polymerase.
  • sequence not involved in physiological activity means a sequence other than the nucleotide sequence necessary for polymerase, particularly a reverse transcriptase-specific action (for example, reverse transcriptase reaction etc.).
  • Primers for amplifying a polymerase, particularly a gene encoding reverse transcriptase hybridize to a sequence other than the sequence part involved in the biological activity more specifically, in order to hybridize to a sequence known per se or the inventors of the present application. It is preferred to use a promoter-splitting primer invented by (see: WO02 / 018586).
  • the “promoter-splitting primer” means “two types of primers (5 ′ end of the promoter) that satisfy the condition that transcription from DNA constructed using only one type of primer as the 5′-side primer does not occur. And a polynucleotide having a sequence complementary to a nucleotide sequence including at least a part of a promoter functional site, and a polynucleotide having a sequence complementary to a nucleotide sequence including at least a part of an RNA polymerase recognition site from the 3 ′ end of the promoter) 3'-side primer ".
  • the individual length and sequence depend on the complexity and temperature of the required gene and ionic strength.
  • the “gene amplification solution” of the present invention means a solution containing an essential component capable of further amplifying a DNA derived from a virus-derived polymerase in a sample, particularly reverse transcriptase RNA, by reverse transcription. To do.
  • the gene amplification solution is a solution for performing PCR, reverse transcriptase, dNTPs, polymerase, and the primers described in the above paragraph are included.
  • NASBA method Nucleic Acid Sequence Based Amplification method, Nature, 350, 91-92, 1991, Patent No. 2648802 and Patent No.
  • the process can be greatly simplified, and a large number of transcription templates can be synthesized in a short time with a small number of steps. It becomes. That is, since a step for preparing a plasmid incorporating a DNA encoding polymerase, particularly reverse transcriptase, is not required, ultracentrifugation for plasmid purification can be omitted.
  • the “transcription solution” of the present invention means a solution containing an essential component for using an amplified polymerase, particularly a reverse transcriptase gene (DNA) as a translation template.
  • a transcription reaction such as RNA polymerase (for example, SP6 RNA polymerase) and a substrate for RNA synthesis (four kinds of ribonucleoside triphosphates).
  • the transcription reaction is carried out by incubating the solution at about 20 ° C. to about 60 ° C., preferably about 30 ° C. to about 42 ° C., for about 30 minutes to about 16 hours, preferably about 2 hours to about 5 hours. Is called.
  • a polymerase particularly a reverse transcriptase
  • a transcription solution containing a polymerase particularly a reverse transcriptase translation template
  • a reverse transcriptase translation template can be easily obtained by adding a transcription solution containing a polymerase, particularly a reverse transcriptase translation template, to the following cell-free protein synthesis system without purification.
  • a transcription solution containing a polymerase particularly a reverse transcriptase translation template
  • the “cell-free protein synthesis system” of the present invention preferably uses an extract for cell-free protein synthesis using eukaryotic-derived wheat germ or the like, and uses this extract to express a polymerase, particularly reverse transcriptase. Means system.
  • examples of commercially available extracts for protein synthesis include Rabbit Reticulocyte Lysate System (Promega) derived from rabbit reticulocytes, Wheat Germ Expression Premium Kit ⁇ WEPRO (TM) ⁇ derived from wheat germ, Cell Free Science Co., Ltd.), etc. .
  • the best extract to be used in the present invention is an extract derived from wheat germ, and an extract from which low molecular substances such as glucose that causes inhibition of protein synthesis in the endosperm components and embryo tissues are further removed. It is a liquid.
  • the extract from which the endosperm component is substantially removed means that the deadenination rate of ribosome is 7% or less, preferably 1% or less.
  • sugar and phosphorylated saccharide are reduced to 10 mM or less, preferably 6 mM or less (as the glucose concentration in the extract having an absorbance of 200 OD / ml at 260 nm).
  • a method for preparing such an extract is exemplified in WO2005 / 063979 A1.
  • the amino acid, energy source, various ions, buffer solution, ATP regeneration, which is a substrate, is added to the cell extract for protein synthesis to which the translation template of the unpurified or purified polymerase (particularly reverse transcriptase) obtained as described above is added.
  • a solution also referred to as a “translation solution”
  • the translation reaction is performed by incubating at an appropriate temperature for an appropriate time.
  • the substrate amino acids are usually 20 types of L-type amino acids constituting the protein, but analogs and isomers thereof can also be used depending on the purpose.
  • ATP and / or GTP are mentioned as an energy source.
  • various ions include acetates such as potassium acetate, magnesium acetate, and ammonium acetate, and glutamates.
  • the nuclease inhibitor include ribonuclease inhibitors and nuclease inhibitors.
  • the reducing agent include dithiothreitol.
  • Antibacterial agents include sodium azide, ampicillin and the like. These addition amounts can be appropriately selected within a range that can be usually used in cell-free protein synthesis.
  • the protein synthesis reaction liquid of the following compositions is preferable.
  • composition of the following final concentration 1,000 units / ml ribonuclease inhibitor (RNasin) (Promega), containing 48% volume of the wheat germ extract described above (concentration is 200 A 260 nm units / ml), 30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 0.5 mg / ml creatine kinase, 1.2 mM adenosine triphosphate (ATP), 0.25 mM guanosine Triphosphate (GTP), 16 mM creatine phosphate, 0.38 mM spermidine, 20 types of L-type amino acids (each 0.3 mM)].
  • Rasin ribonuclease inhibitor
  • the mode of addition of the translation solution can be appropriately selected according to the translation reaction system to be used.
  • the synthesis system used in the method of the present invention may be any method known per se that can be applied to the cell-free protein synthesis method.
  • the batch method ⁇ Pratt, J. M. "Coupled Transcription-Translation in Prokaryotic Cell-Free Systems: in Transcription and Translation, 179-209, Hames, B. D. & Higgins, S. J., eds, IRL Press, Oxford (1984) ⁇ and the multi-layer method (WO02 / 24939) Can be mentioned.
  • Detecting the inhibitory activity of a polymerase inhibitor (reverse transcriptase inhibitor) on a polymerase (particularly reverse transcriptase) means that the polymerase gene (particularly reverse transcriptase gene) derived from each patient is mutated.
  • the pharmacological effects of polymerase inhibitors (reverse transcriptase inhibitors) against mutant polymerases derived from it (mutant reverse transcriptase) may be directly or indirectly affected. means.
  • the HIV reverse transcriptase inhibitor inhibits the nucleotide uptake of HIV reverse transcriptase from each patient. It is to check whether or not.
  • the method for detecting the inhibitory activity described above is that a polymerase (particularly reverse transcriptase) derived from each patient and a wild type polymerase (particularly wild type reverse transcriptase) in the presence of the polymerase inhibitor (reverse transcriptase inhibitor). ) To compare the physiological activity. Specific examples are as described below.
  • Method for detecting the inhibitory activity of a nucleic acid-based or non-nucleic acid-based reverse transcriptase inhibitor against reverse transcriptase (1) At least one 5 ′ primer and one 3 ′ primer that specifically hybridize to a sequence other than the sequence part involved in the physiological activity of reverse transcriptase (eg, HIV-1 reverse transcriptase)
  • a solution containing a primer / template hybrid containing a labeling substance prepared in advance is added to the plate. Furthermore, preferably ATP is added to the plate. Nucleotides containing a labeling substance, acceptor beads capable of directly or indirectly recognizing the labeling substance of the primer or the labeling substance of the nucleotide, and directly or indirectly the labeling substance of the primer or the labeling substance of the nucleotide Recognizable donor beads are added to the plate simultaneously or separately.
  • the activity detection method includes a homogeneous assay or a heterogeneous assay.
  • the homogeneous assay is more preferable because the washing step can be omitted.
  • the homogeneous assay is selected from any one or more of the following. 1) ALPHA, 2) surface plasmon resonance method, 3) fluorescence correlation analysis method, 4) fluorescence intensity distribution analysis method, 5) FRET, 6) BRET, 7) EFC, 8) FP
  • the heterogeneous assay is selected from any one or more of the following. 1) ELISA, 2) DELFIA, 3) SPA, 4) Flash plate analysis
  • FRET Fluorescence Resonance Energy Transfer
  • ALPHA Analog to Physical Reduction Agent
  • ALPHA Analog to Physical Reduction Agent
  • the method is an analytical method based on the movement of singlet oxygen between a donor bead and an acceptor bead brought into close proximity. This is because upon excitation at 680 nm, the photosensitizer in the donor bead converts ambient oxygen to singlet state oxygen, which diffuses to a distance of 200 nm. The chemiluminescent group in the acceptor bead transfers energy to the fluorescent acceptor in the bead and subsequently emits light at a wavelength of about 600 nm.
  • the acceptor beads are inert carriers such as glass, silica gel, and resin, and are carriers for immobilizing the biomolecules.
  • Donor beads are inert carriers such as glass, silica gel, and resin, and are carriers for immobilizing streptavidin.
  • a reverse transcriptase, one or a plurality of reverse transcriptase inhibitors, a template, a primer containing biotin as a labeling substance, a nucleotide containing DIG as the labeling substance, acceptor beads capable of directly or indirectly recognizing the DIG, and Avidin or streptavidin capable of directly or indirectly recognizing the biotin is introduced in vitro into donor beads.
  • ATP is preferably added in vitro.
  • the reverse transcriptase inhibitor cannot inhibit the reverse transcriptase activity due to the mutation of the reverse transcriptase, the reverse transcription reaction does not stop (see: lower diagram in FIG. 2). Therefore, as shown in the lower diagram of FIG. 2, the donor beads and the acceptor beads are close to each other, and the signal rises.
  • the reverse transcriptase inhibitor inhibits the reverse transcriptase activity, the reverse transcription reaction stops (see: upper diagram in FIG. 2). Therefore, the signal does not increase because the donor beads and the acceptor beads are not close as shown in the upper diagram of FIG. Signal detection is performed, for example, by measuring the fluorescence intensity emitted from the acceptor beads.
  • Reverse transcriptase one or more reverse transcriptase inhibitors, template, primer containing fluorescent substance as labeling substance, nucleotide containing quenching substance as labeling substance, or reverse transcriptase, one or more A reverse transcriptase inhibitor, a template, a primer containing a quenching substance as a labeling substance, and a nucleotide containing a fluorescent substance as a labeling substance are introduced in vitro.
  • ATP is preferably added in vitro.
  • the reverse transcriptase inhibitor when the reverse transcriptase inhibitor does not inhibit the reverse transcriptase activity due to the mutation of the reverse transcriptase, the reverse transcription reaction does not stop. Therefore, since the fluorescent substance and the quenching substance (quencher) are close to each other and the fluorescence energy emitted from the fluorescent substance is absorbed by the quencher, the fluorescence is attenuated (the signal rise is suppressed). On the other hand, when the reverse transcriptase inhibitor inhibits the reverse transcriptase activity, the reverse transcription reaction stops. Therefore, the fluorescent substance and the quenching substance (quencher) cannot be brought close to each other, the distance between the fluorescent substance and the quencher is increased, and the fluorescent energy cannot be absorbed.
  • the fluorescent substance emits fluorescence of its intrinsic wavelength (an increase in signal occurs).
  • the combination of the fluorescent substance and the quencher that effectively attenuates the fluorescence is not particularly limited as long as it has such a function.
  • fluorescent substances such as fluorescein, FITC, FAM, TAMRA, Cy3, Cy5, europium, and the like, emit longer wavelength fluorescence that has an absorption wavelength that matches or is close to the fluorescence wavelength.
  • Another fluorescent substance can be combined as a quencher.
  • a non-fluorescent substance that releases absorbed energy as heat instead of fluorescence can be used as a quencher. These are called dark quenchers, and many have excellent fluorescence attenuation efficiency.
  • Dabcyl, Eclipse, and BHQ Black Hole Quencher
  • the “method for selecting a reverse transcriptase inhibitor suitable for a patient” of the present invention is performed based on the signal detection result. Specifically, the above signal detection is performed using each HIV reverse transcriptase inhibitor, and drug resistance profiling as shown in FIG. 7 is created. Thereby, drug resistance peculiar to each patient can be specified, and one or a plurality of HIV reverse transcriptase inhibitors suitable for each patient can be selected. In this example, the reaction of only one inhibitor per sample is detected, but it is also possible to detect the additive / synergistic effect of a plurality of inhibitors per sample.
  • the “kit for measuring the inhibitory activity of a reverse transcriptase inhibitor” of the present invention includes at least a primer containing a template and a labeling substance.
  • ATP is added so as to be about 0.5 mM to 15 mM with respect to the reaction system.
  • the TAM mutation means the amino acid mutation at positions 41, 67, 70, 210, 215, and 219 of HIV reverse transcriptase.
  • HIV-1 reverse transcriptase was synthesized in a wheat germ cell-free protein synthesis system. Details are as follows.
  • the reverse transcriptase HIV-RT ⁇ HIV-1 reverse transcriptase (p51, p66) in a 1: 1 ratio ⁇ DNA template solution is added to the transfer solution ⁇ 30 ⁇ l of 5x transcription buffer (400 mM HEPES, pH 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT), 15 ⁇ l of 25 mM 4NTPs, 1.875 ⁇ l of RNasin (80 Units), 1.875 ⁇ l of SP6 polymerase (80 units), 71.25 ⁇ l water ⁇
  • a translation template was prepared by performing a transcription reaction at 37 ° C. for 3 hours. Further, the solution containing the translation template was added to a wheat germ cell-free protein synthesis system, and protein synthesis was performed at 16 ° C. for 15 to 20 hours to obtain HIV-RT.
  • HIV-RT (WT: wild type): synthesized by the method described in Example 1.
  • Substrate solution Biotin-16-dUTP (1 ⁇ M), dTTP (1 ⁇ M), dATP (10 ⁇ M), dGTP (10 ⁇ M), dCTP (10 ⁇ M) in 50 mM Tris-HCl (pH 7.8)
  • Incubation buffer 50 mM Tris-HCl (pH 7.8), 319 mM potassium chloride, 33 mM magnesium chloride, 11 mM DTT Template: RNA3 (SEQ ID NO: 1) or RNA4 (SEQ ID NO: 2)
  • Primer Primer1 (without labeling substance: TGGATCTTGGAGTTC: SEQ ID NO: 3) or Primer1 containing labeling substance ⁇ with labeling substance: DIG-TGGATCTTGGAGTTC (SEQ ID NO: 3) ⁇ Solution containing Template / primer hybrid: RNA3 or RNA4 (12.5 pmol / ⁇ l) and
  • the signal value of the reverse transcriptase activity in the measurement system using the primer containing the labeling substance is 2 in comparison with the signal value in the measurement system using the primer not containing the labeling substance. More than 3 times.
  • This result shows that reverse transcriptase activity can be measured with high sensitivity by using “a primer containing a labeling substance”, which is one of the characteristics of the measurement method of the present invention.
  • a primer containing a labeling substance which is one of the characteristics of the measurement method of the present invention.
  • RNA4 was used as a template and showed a higher signal value as compared with the case of using RNA3, it can be seen that RNA4 is a more preferable template than RNA3.
  • HIV-RT (WT: wild type): synthesized by the method described in Example 1.
  • Substrate solution Biotin-16-dUTP (1 ⁇ M), dTTP (0 ⁇ M or 1 ⁇ M), dATP (10 ⁇ M), dGTP (10 ⁇ M), dCTP (10 ⁇ M) in 50 mM Tris-HCl (pH 7.8)
  • Incubation buffer 50 mM Tris-HCl (pH 7.8), 319 mM potassium chloride, 33 mM magnesium chloride, 11 mM DTT Template: RNA4 (SEQ ID NO: 2)
  • Primer Primer1 containing labeling substance Solution containing Template / primer hybrid: RNA4 (50 pmol / ⁇ l) and Primer1 (50 pmol / ⁇ l) containing a labeling substance were mixed in equal amounts and reacted at 65 ° C.
  • Reaction buffer 50 mM Tris-HCl (pH 7.8), 80 mM potassium chloride, 2.5 mM DTT, 0.75 mM EDTA, 0.5% Triton X-100 AlphaScreen (TM)
  • DIG detection kit No.6760604C (PerkinElmer) Beads (Beads mix): An equivalent mixture of Streptavidin Donor Beads and anti-Digoxin / Digoxigenin (DIG) Acceptor Beads was diluted 100-fold with 1 mg BSA / ml PBS.
  • the detection sensitivity of the reverse transcriptase activity was improved by lowering the concentration of the template / primer hybrid in the reverse transcriptase activity measurement solution. Furthermore, the S (signal) / N (noise) ratio also increased to about 10 times. Thereby, in the following Examples, the density
  • HIV-RT (Materials used) HIV-RT (WT: wild type): synthesized by the method described in Example 1.
  • Nucleic acid inhibitors AZTTP (1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM), 3TCTP (1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM), TDFDP (1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM)
  • Non-nucleic acid inhibitors EFV (1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM), NVP (1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM)
  • Substrate solution Biotin-16-dUTP (500 nM), dTTP (500 nM), dATP (1 ⁇ M), dGTP (1 ⁇ M), dCTP (1 ⁇ M) in 50
  • the measurement results are shown in FIG. In FIG. 5, the signal value at each concentration is converted to% with the signal value when no reverse transcriptase inhibitor is added as 100. As is clear from the results in FIG. 5, various reverse transcriptase inhibitors inhibited HIV reverse transcriptase activity in a concentration-dependent manner. That is, by using the measurement method of the present invention, it was shown that the reverse transcriptase inhibitory activity can be measured with one type of template regardless of whether it is a nucleic acid or non-nucleic acid.
  • TDF a reverse transcriptase inhibitor
  • TCCCCCCCCC SEQ ID NO: 6
  • RNA5-C AAAAAAAAAAAAAAACCCCCCCCCCCCGAACUCCAAGAUCCA: SEQ ID NO: 5
  • dATP and TDFDP are not used as HIV reverse transcriptase substrates, so TDFDP is HIV Does not show reverse transcriptase inhibitory activity. Details are as follows.
  • HIV-RT (WT: wild type): synthesized by the method described in Example 1.
  • Nucleic acid inhibitors TDFDP (1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM)
  • Substrate solution Biotin-16-dUTP (500 nM), dTTP (500 nM), dATP (1 ⁇ M), dGTP (1 ⁇ M), dCTP (1 ⁇ M) in 50 mM Tris-HCl (pH 7.8)
  • Incubation buffer 50 mM Tris-HCl (pH 7.8), 319 mM potassium chloride, 33 mM magnesium chloride, 11 mM DTT Template: RNA4 (SEQ ID NO: 2), RNA5 (SEQ ID NO: 4), RNA5-C (SEQ ID NO: 5)
  • Primer Primer1 with labeling substance Solution containing Template / primer hybrid: RNA4, RNA5 or RNA5-C (5 pmol / ⁇ l
  • the measurement results are shown in FIG. In FIG. 6, the signal value at each concentration is converted to% with the signal value when no reverse transcriptase inhibitor is added as 100.
  • RNA4 or RNA5 was used as a template
  • HIV reverse transcriptase activity was inhibited depending on the concentration of the reverse transcriptase inhibitor.
  • RNA5-C was used as a template
  • the inhibitory activity of the reverse transcriptase inhibitor was not confirmed.
  • the reverse transcriptase recognizes the inhibitor complementary sequence of the template and the reverse transcriptase inhibitor inhibits the reverse transcriptase activity at the inhibitor complementary sequence site. confirmed.
  • the signal value in the measurement system using RNA5 as a template was higher than the signal value when RNA4 was used (data not shown). Therefore, it was confirmed that RNA5 is a more preferable template than RNA4.
  • HIV-RT NL4-3 HIV-RT derived from HIV: synthesized by the method described in Example 1.
  • Inhibitor 3TCTP (0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M, 1000 ⁇ M) : AZTTP (1nM, 10nM, 100nM, 1000nM, 10000nM) : TDFDP (10nM, 100nM, 1000nM, 10000nM, 100000nM) : EFV (10nM, 100nM, 1000nM, 10000nM, 100000nM)
  • Substrate solution Biotin-16-dUTP (500 nM), dTTP (500 nM), dATP (1 ⁇ M), dGTP (1 ⁇ M), dCTP (1 ⁇ M) in 50 mM Tris-HCl (pH 7.8)
  • Incubation buffer 50 mM Tris-HCl (pH 7.8), 319 mM potassium chloride, 33 mM magnesium chloride, 11 mM DTT Template: RNA5 (SEQ ID NO: 4)
  • Primer Primer1 with labeling substance Solution containing Template
  • FIG. 8 shows the inhibitor 3TCTP for mutant HIV1-RT ⁇ HIV-1 p7324-4 (No. 2), HIV-1 p7303-3 (No. 6), HIV-1 p56252-1 (No. 10) ⁇ . It shows the inhibitory activity.
  • the signal value at each concentration is converted to%, where the signal value when no reverse transcriptase inhibitor is added is 100.
  • FIG. 7 shows that the resistance to 3TC, an inhibitor, is HIV-1 p56252-1 (No. 10), HIV-1 p7303-3 (No. 6), and HIV-1 p7324-4 (No. 2). Indicates that it is in order.
  • HIV-1 p56252-1 No. 10
  • HIV-1 p7303-3 No. 6
  • HIV-1 p7324-4 No. 2 It was in order. That is, the measurement results using the present invention are consistent with the data predicted by the Stanford drug resistance database.
  • FIG. 9 shows the results of using inhibitors AZTTP, TDFDP, or EFV for mutant HIV1-RT ⁇ HIV-1 p1617-1 (No. 7), HIV p56252-1 (No. 10) ⁇ .
  • FIG. 7 shows that HIV-1 p1617-1 (No. 7) and HIV-1 p56252-1 (No. 10) have high resistance to AZT.
  • HIV-1 p1617-1 (No. 7) and HIV-1 p56252-1 (No. 10) were hardly inhibited by reverse transcriptase activity even at high concentrations of AZT.
  • FIG. 7 shows that HIV-1 p56252-1 (No.
  • HIV-1 p56252-1 (No. 10) has a higher resistance to TDF than HIV-1 p1617-1 (No. 7).
  • HIV-1 p56252-1 (No. 10) showed higher reverse transcriptase activity than HIV-1 p1617-1 (No. 7).
  • FIG. 7 shows that HIV-1 p1617-1 (No. 7) has no resistance to EFV, and HIV-1 p56252-1 (No. 10) has a high resistance to EFV.
  • HIV-1 p1617-1 (No. 7) shows almost no reverse transcriptase activity, whereas HIV-1 p56252-1 (No. 10) has a high reverse transcriptase activity. Showed activity. That is, the measurement results using the present invention agreed with the data predicted by the Stanford drug resistance database.
  • NL4-3 HIV-RT derived from HIV synthesized by the method described in Example 1.
  • AZT resistant HIV1-RT ⁇ HIV-1 p7303-3 (No. 6) ⁇ synthesized by the method described in Example 1.
  • Inhibitor AZTTP (1nM, 10nM, 100nM, 1000nM, 10000nM) ATP added: 0 mM (control), 5 mM Substrate solution: Biotin-16-dUTP (500 nM), dTTP (500 nM), dATP (1 ⁇ M), dGTP (1 ⁇ M), dCTP (1 ⁇ M) in 50 mM Tris-HCl (pH 7.8) Incubation buffer: 50 mM Tris-HCl (pH 7.8), 319 mM potassium chloride, 33 mM magnesium chloride, 11 mM DTT Template: RNA5 (SEQ ID NO: 4) Primer: Primer1 with labeling substance Solution containing Template / primer hybrid: RNA5 (5 pmol / ⁇ l) and Primer1 (5 pmol / ⁇ l) containing a labeling substance were mixed in equal amounts and reacted at 65 ° C.
  • the measurement results are shown in FIG.
  • the upper part of FIG. 10 shows the inhibitory activity of the inhibitor AZT (TP) against mutant HIV1-RT ⁇ HIV-1 p7303-3 (No. 6) ⁇ .
  • the signal value at each concentration is converted into%, where the signal value when no reverse transcriptase inhibitor is not added is 100.
  • the lower part of FIG. 10 shows the detected value of IC50 (nM).
  • IC50 nM
  • the method for measuring reverse transcriptase activity using the template and primer of the present invention can accurately measure the degree of drug resistance of reverse transcriptase having resistance to each reverse transcriptase inhibitor. Indicated.
  • the above results show that by using the measurement method of the present invention, it is possible to easily measure how much resistance the reverse transcriptase obtained from each patient has to which reverse transcriptase inhibitor, It shows that ATP addition can improve the detection sensitivity of the drug sensitivity of reverse transcriptase.
  • a reverse transcriptase inhibitor suitable for each patient can be selected based on the measurement results.
  • the present invention provides a simple, rapid and reliable method for measuring the inhibitory activity of a reverse transcriptase inhibitor.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
PCT/JP2013/065299 2012-06-04 2013-06-03 Procédé de mesure de l'activité inhibitrice d'un inhibiteur de polymérase Ceased WO2013183572A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012-126648 2012-06-04
JP2012126648A JP2015156805A (ja) 2012-06-04 2012-06-04 ポリメラーゼ阻害剤の阻害活性の測定方法

Publications (1)

Publication Number Publication Date
WO2013183572A1 true WO2013183572A1 (fr) 2013-12-12

Family

ID=49711964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/065299 Ceased WO2013183572A1 (fr) 2012-06-04 2013-06-03 Procédé de mesure de l'activité inhibitrice d'un inhibiteur de polymérase

Country Status (2)

Country Link
JP (1) JP2015156805A (fr)
WO (1) WO2013183572A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009150845A1 (fr) * 2008-06-12 2009-12-17 株式会社セルフリーサイエンス Procédé de sélection d'un agent antiviral le mieux approprié

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009150845A1 (fr) * 2008-06-12 2009-12-17 株式会社セルフリーサイエンス Procédé de sélection d'un agent antiviral le mieux approprié

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
WANG YING-KAI ET AL.: "A homogeneous, solid- phase assay for hepatitis C virus RNA-dependent RNA polymerase", ANALYTICAL BIOCHEMISTRY, vol. 359, 2006, pages 106 - 111 *
YU L. ET AL.: "Fluorescence-Based, High- Throughput DNA Polymerase Assay", BIOTECHNIQUES, vol. 33, 2002, pages 938 - 941 *
ZHANG JI-HU ET AL.: "A High-Throughput Homogeneous Assay for Reverse Transcriptase Using Generic Reagents and Time-Resolved Fluorescence Detection", ANALYTICAL BIOCHEMISTRY, vol. 281, 2000, pages 182 - 186 *

Also Published As

Publication number Publication date
JP2015156805A (ja) 2015-09-03

Similar Documents

Publication Publication Date Title
CN111394430B (zh) 一种基于CRISPR-Cas12a耦合增强型链置换扩增的检测系统及其应用
Jochmans et al. Indolopyridones inhibit human immunodeficiency virus reverse transcriptase with a novel mechanism of action
US12410488B2 (en) Method for detecting coronavirus (SARS-CoV-2)
Jockusch et al. Triphosphates of the two components in DESCOVY and TRUVADA are inhibitors of the SARS-CoV-2 polymerase
JP2009000063A (ja) 改良されたノロウイルスｒｎａ検出方法
JP2019013248A (ja) Ｈｅｖ核酸を検出するための組成物および方法
US7399588B2 (en) Method for detecting SARS coronavirus
JP2022501073A (ja) 水痘帯状疱疹ウイルスを増幅または検出するための組成物および方法
WO2009150845A1 (fr) Procédé de sélection d'un agent antiviral le mieux approprié
US10689689B2 (en) Generic method for the stabilization of specific RNA
JP6409233B2 (ja) キットおよび方法
WO2013183572A1 (fr) Procédé de mesure de l'activité inhibitrice d'un inhibiteur de polymérase
JP6733215B2 (ja) プライマー及びマイコプラズマ・ニューモニエの検出方法
ES2759991T3 (es) Ensayo para detectar y cuantificar el VIH-1
Xu et al. A T7 exonuclease-RCA dual amplification system for high-sensitivity and high-selectivity analysis of microRNA
ES2336807T3 (es) Deteccion del tipo y subtipo del vih.
US20050170338A1 (en) Norovirus detection reagent
JP2022105717A (ja) アデノウイルス、メタニューモウイルス、および／またはライノウイルス核酸を検出するための組成物、方法、およびキット
JP4748686B2 (ja) 逆転写酵素のｈ型ｒｎａ分解酵素阻害剤のスクリーニング方法
US12110539B2 (en) Metal ion-start DNA polymerase switch and isothermal polymerase amplification method using the same
US20230416849A1 (en) Methods for detecting viral rna using atp-releasing nucleotides
US9157128B2 (en) Kit for detecting HIV-2 and method for detecting HIV-2 using the same
Xiao et al. The 2’-endo conformation of arabinose-CTP and arabinose-UTP inhibit viral polymerases by inducing long pauses
JP6593984B2 (ja) Ｒｎａ増幅法およびｈｉｖ−１感染診断方法
JP2007135469A (ja) 核酸増幅方法

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: 13800720

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: 13800720

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP