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WO2013147043A1 - Procédé de détection ou de dosage d'acide nucléique et kit de réactifs afférent - Google Patents

Procédé de détection ou de dosage d'acide nucléique et kit de réactifs afférent Download PDF

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Publication number
WO2013147043A1
WO2013147043A1 PCT/JP2013/059307 JP2013059307W WO2013147043A1 WO 2013147043 A1 WO2013147043 A1 WO 2013147043A1 JP 2013059307 W JP2013059307 W JP 2013059307W WO 2013147043 A1 WO2013147043 A1 WO 2013147043A1
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Prior art keywords
nucleic acid
target nucleic
reagent
test sample
egta
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PCT/JP2013/059307
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English (en)
Japanese (ja)
Inventor
奈穂 千葉
田中　和生
力 幸田
水野　克己
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株式会社シノテスト
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Priority to JP2014508041A priority Critical patent/JP6402347B2/ja
Publication of WO2013147043A1 publication Critical patent/WO2013147043A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a nucleic acid detection or quantification method and a detection or quantification reagent kit.
  • a method for detecting a nucleic acid contained in a biological sample such as blood a method using a nucleic acid amplification reaction such as PCR (polymerase chain reaction) is known.
  • PCR polymerase chain reaction
  • a method of isolating or purifying nucleic acid from a biological sample is generally used as a pretreatment for the nucleic acid amplification reaction.
  • a method in which a biological sample is directly used for a nucleic acid amplification reaction without pretreatment such as isolation and purification of nucleic acid has been developed.
  • Patent Document 1 a method using a reaction mixture containing a zwitterionic buffer and / or a non-reducing carbohydrate (Patent Document 1), characterized in that the pH of the PCR reaction solution is in a specific range.
  • Patent Document 2 a method characterized by performing a heating treatment or a treatment with a glycolytic enzyme (Patent Document 3), and determining the concentration of positive monovalent ions and divalent ions in a reaction mixture
  • Patent Document 4 a method characterized by setting the range is known.
  • the present invention has been made in view of such a situation, and the problem to be solved is to detect a target nucleic acid contained in the test sample quickly and accurately without purifying the nucleic acid from the test sample.
  • An object of the present invention is to provide a novel nucleic acid detection or quantification reagent kit and detection or quantification method that can be quantified.
  • ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA) or 1,2 -Bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA), oligonucleotide for amplifying the target nucleic acid and test sample are mixed, and the mixture is subjected to nucleic acid amplification reaction.
  • a reagent kit for detecting or quantifying a target nucleic acid which is ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis ( o-aminophenoxy) ethane-N, N, N ′, N′-tetraacetic acid (BAPTA) and at least one compound selected from the group consisting of these and salts thereof, and an oligo for amplifying the target nucleic acid
  • the reagent kit comprising a nucleotide.
  • EGTA Ethylene glycol-bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid
  • BAPTA 1,2-bis (o-aminophenoxy) ethane-N, N, N' , N′-tetraacetic acid
  • the reagent kit according to (1) above comprising: (3) The reagent kit according to (1) or (2) above, wherein the target nu
  • the microorganism is a virus or a bacterium.
  • a method for detecting or quantifying a target nucleic acid comprising ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis (o -Aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA) and at least one compound selected from the group consisting of these derivatives, or a salt thereof, a test sample, and the target nucleic acid is amplified.
  • EGTA ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid
  • BAPTA 1,2-bis (o -Aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid
  • a method for detecting or quantifying the amplified target nucleic acid by mixing the target oligonucleotide with a target oligonucleotide, performing an amplification reaction of the target nucleic acid in the mixture.
  • a method for detecting or quantifying a target nucleic acid comprising the following steps.
  • step (a) Ethylene glycol-bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane-N, N, N' , N′-tetraacetic acid (BAPTA) and a first reagent containing at least one compound selected from the group consisting of these derivatives or a salt thereof, a test sample, and a target nucleic acid derived from a virus or bacteria Mixing with a second reagent comprising an oligonucleotide; and (b) A step of performing an amplification reaction of the target nucleic acid in the mixture obtained in step (a) and detecting or quantifying the amplified target nucleic acid
  • the target nucleic acid contained in the test sample can be detected or quantified quickly and accurately without purifying the nucleic acid from the test sample.
  • the present invention relates to ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA), a derivative thereof or a salt thereof, and an oligonucleotide for amplifying a target nucleic acid and a test sample are mixed, a nucleic acid amplification reaction is performed using the mixture, and the test is performed.
  • the present invention relates to a method for detecting or quantifying a target nucleic acid contained in a sample, and a reagent kit that can be used in the method.
  • a biological sample has a substance or property that inhibits a nucleic acid amplification reaction. Therefore, when a target nucleic acid contained in a biological sample is amplified and detected or quantified by a nucleic acid amplification reaction, the nucleic acid contained in the biological sample is purified. The method is adopted. On the other hand, when a target nucleic acid contained in a biological sample is amplified and detected or quantified by a nucleic acid amplification reaction without purifying the nucleic acid from the biological sample, a factor that inhibits the nucleic acid amplification reaction in the biological sample is determined by some method. It is necessary to suppress it.
  • the present invention provides a reagent kit for detecting or quantifying a target nucleic acid, comprising at least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof, or a salt thereof, and an oligonucleotide for amplifying the target nucleic acid.
  • a reagent kit for detecting or quantifying a target nucleic acid comprising at least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof, or a salt thereof, and an oligonucleotide for amplifying the target nucleic acid.
  • the present invention also relates to a method for detecting or quantifying a target nucleic acid for amplifying at least one compound selected from the group consisting of EGTA, BAPTA and derivatives thereof, a salt thereof, a test sample, and the target nucleic acid. And a method for detecting or quantifying the amplified target nucleic acid by carrying out an amplification reaction of the target nucleic acid in the mixture.
  • the present invention also provides a method for detecting or quantifying a target nucleic acid, comprising: (a) a first reagent containing at least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof, or a salt thereof; The sample was mixed with a second reagent containing an oligonucleotide for amplifying the target nucleic acid, and (b) the target nucleic acid in the mixture obtained by the step (a) was amplified and amplified.
  • the method is provided comprising the step of detecting or quantifying the target nucleic acid.
  • the target nucleic acid contained in the test sample can be detected or quantified quickly and accurately without purifying the nucleic acid from the test sample.
  • target nucleic acid refers to a nucleic acid to be amplified, detected or quantified
  • target nucleic acid sequence refers to a base sequence of the target nucleic acid.
  • the target nucleic acid can be appropriately selected according to the purpose of detection or quantification, but is preferably DNA or RNA derived from a microorganism.
  • DNA includes total DNA, cDNA, genomic DNA and synthetic DNA.
  • RNA includes mRNA, rRNA, genomic RNA, and synthetic RNA.
  • the microorganism from which the target nucleic acid is derived is not limited as long as it has a nucleic acid genome, but a microorganism having pathogenicity is preferred.
  • examples of the microorganism include viruses, bacteria, fungi, yeasts, and slime molds, and viruses and bacteria are preferable.
  • viruses include DNA viruses having DNA as a genome and RNA viruses having RNA as a genome, with DNA viruses being preferred.
  • the DNA virus includes a double-stranded DNA virus and a single-stranded DNA virus, and preferably a double-stranded virus.
  • examples of the double-stranded DNA virus include herpes virus, adenovirus, pox virus, etc. Herpes virus is preferred.
  • Herpes virus is a virus whose host is an animal, and has been found in many mammals, birds, amphibians, reptiles, fish and the like.
  • a herpesvirus having a human host is preferred.
  • human herpes virus include, but are not limited to, human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus.
  • VSV Epstein-Barr virus
  • EBV human herpes virus 6
  • HHV-7 human herpes virus 7
  • KSHV Kaposi's sarcoma-related herpes virus
  • HCMV human cytomegalovirus
  • HSV-1 herpes simplex virus type 1
  • HCMV Human herpesviruses are responsible for various diseases.
  • HCMV is involved in HCMV infection, interstitial pneumonia, CMV retinopathy, CMV mononucleosis, congenital giant cell inclusion disease
  • HSV-1 is cold sores, genital herpes, Kaposi varicella-like rash, Involved in herpes encephalitis, corneal herpes, bell paralysis.
  • HSV-2 is involved in genital herpes, neonatal herpes, myelitis, aseptic meningitis, and acute retinal necrosis
  • VSV is involved in chickenpox, herpes zoster, and Ramsey-Hunt syndrome.
  • EBV is involved in infectious mononucleosis, chronic active EBV infection, nasopharyngeal cancer, Burkitt lymphoma, EBV-related gastric cancer, and HHV-6 is involved in sudden rash, encephalitis / encephalopathy . Furthermore, HHV-7 is involved in idiopathic rash, and HHV-8 is involved in Kaposi's sarcoma (AIDS-related, classical, African), Castleman's disease, and malignant B lymphoma.
  • Kaposi's sarcoma AIDS-related, classical, African
  • Castleman's disease and malignant B lymphoma.
  • RNA viruses include parvovirus (such as adeno-associated virus).
  • RNA viruses include double-stranded RNA viruses and single-stranded RNA viruses.
  • Single-stranded RNA viruses include retroviruses (RNA tumor viruses, human immunodeficiency viruses, etc.), rhabdoviruses (rabies virus, vesicular stomatitis virus, etc.), paramyxoviruses (Sendai virus, mumps virus, measles virus, etc.) , Orthomyxovirus (such as influenza virus), arena virus (such as lymphocytic choriomeningitis virus, lassa virus), coronavirus (such as SARS (Severe Acute Respiratory Syndrome) virus), norovirus and the like.
  • retroviruses RNA tumor viruses, human immunodeficiency viruses, etc.
  • rhabdoviruses rabies virus, vesicular stomatitis virus, etc.
  • any region of virus-derived DNA or RNA can be selected.
  • a conserved region of DNA or RNA with few gene mutations can be selected as the target nucleic acid.
  • DNA containing a base sequence such as glycoprotein B gene (SEQ ID NO: 1), glycoprotein H gene (SEQ ID NO: 2), matrix phosphorylated protein pp65 gene (SEQ ID NO: 3) is used as a target nucleic acid.
  • SEQ ID NO: 3 a base sequence
  • Such gene information can be obtained from a known gene information database (for example, ENA of European Bioinformatics Institute (EBI)).
  • the bacterium includes both gram positive bacteria and gram negative bacteria.
  • Gram-positive bacteria include Staphylococcus aureus and other Staphylococcus bacteria, Streptococcus bacteria, Listeria ⁇ ⁇ ⁇ monocytogenes and other Listeria genus Bacillus cereus and other Bacillus cereus Examples include bacteria, Mycobacterium bacteria such as Mycobacterium tuberculosis, Mycoplasma bacteria, Clostridium botulinum, Clostridium bacteria such as Clostridium perfringens, and the like.
  • Gram-negative bacteria include Escherichia bacteria such as Escherichia coli, Citrobacter bacteria such as Citrobacter koseri, Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia pneumoniae. ), Chlamydia bacteria such as Klebsiella oxytoca, Enterobacteriaceae represented by Klebsiella bacterium such as Klebsiella oxytoca, Vibrio bacterium such as Vibrio cholerae, and Haemophilus genus Haemophilus influenzae Examples include bacteria, Salmonella bacteria, Proteus bacteria, Pseudomonas bacteria, Neisseria gonorrhoeae and the like.
  • an arbitrary region can be selected as a target nucleic acid in DNA derived from bacteria.
  • a region containing a gene that causes a disease can be selected as a target nucleic acid.
  • virulence genes include Listeria ricin O (hlyA) gene of Listeria bacterium, enterotoxin gene and invasion (invA) gene of Salmonella bacterium, verotoxin gene of pathogenic E. coli O-157, Examples include the Staphylococcus aureus enterotoxin gene, Bacillus cereus cereulide (vomiting toxin) gene, enterotoxin gene, and botulinum toxin genes.
  • a target nucleic acid derived from a microorganism can be selected from any region of microorganism-derived DNA or RNA based on known gene information and a gene database (for example, NCBI GenBank), and a primer capable of amplifying the region.
  • the nucleic acid sequence to be amplified may be either the full length or a part of the target nucleic acid sequence.
  • DNA containing the base sequence of glycoprotein H gene of human cytomegalovirus is selected as a target nucleic acid, and an oligonucleotide primer that can amplify all or part of the target nucleic acid is used.
  • the target nucleic acid can be amplified and detected or quantified by a nucleic acid amplification reaction (Eiko Fukushima, et al., Journal of Virological Methods 151 (2008) 55-60).
  • a person skilled in the art can detect or quantify other nucleic acid-derived target nucleic acids using the same technique.
  • the base sequence of the target nucleic acid to be detected or quantified may be, for example, a base sequence on the sense side or a base sequence on the antisense side as long as it is a double-stranded nucleic acid.
  • a complementary nucleic acid sequence can be detected or quantified by detecting or quantifying the antisense nucleic acid sequence.
  • the “test sample” means a sample to be examined for the presence or amount of the target nucleic acid, and examples of the target nucleic acid include those contained in the test sample.
  • the test sample may be a biological sample, a food sample, a pharmaceutical product, a sample of natural water (hereinafter referred to as “biological sample etc.”) itself, or a diluted or concentrated sample. That is, as the test sample, one containing a biological sample or the like can be used. Further, the test sample may include other components, microorganisms, solvents and the like in addition to a sample such as a biological sample.
  • target nucleic acid contained in a test sample and “target nucleic acid contained in a biological sample” represent the same meaning.
  • the biological sample refers to a sample derived from a living body, for example, a body fluid sample, mucosa (oral mucosa, gastric mucosa, airway mucosa) and cells thereof, feces, skin, other cells (including cultured cells), A part of each tissue may be mentioned, and a body fluid sample is preferable.
  • a body fluid sample for example, a body fluid sample, mucosa (oral mucosa, gastric mucosa, airway mucosa) and cells thereof, feces, skin, other cells (including cultured cells), A part of each tissue may be mentioned, and a body fluid sample is preferable.
  • a body fluid sample refers to a sample containing a body fluid derived from a living body, such as urine, saliva, amniotic fluid, breast milk, blood (whole blood, serum, plasma), spinal fluid, synovial fluid, ascites, pleural effusion, ear leakage, nasal discharge, pus, Examples include bile, sputum, sweat, and other pulverized liquids of cells or tissues.
  • the body fluid sample is preferably selected from the group consisting of urine, saliva, amniotic fluid, breast milk, serum and plasma, and more preferably selected from the group consisting of urine, saliva, amniotic fluid and breast milk.
  • the body fluid sample may contain cells.
  • the living body includes individual animals, animal tissues, animal cells (including cultured cells), and the like.
  • animals include humans, mice, rats, horses, dogs, sheep, rabbits, cows, pigs, rhesus monkeys, common marmosets, chickens, etc., preferably humans, mice, rats, more preferably humans. It is.
  • the age of the target human is not limited, and is appropriately selected within the range from the fetal period to 120 years depending on the purpose of detection.
  • cytomegalovirus infection when diagnosing or detecting congenital cytomegalovirus infection, preferably within 21 days of life from the fetal period, and when diagnosing or detecting acquired cytomegalovirus infection, from 1 month after birth 12 years old is preferable, and when detecting or diagnosing cytomegalovirus infection in an immunodeficient human, it is preferable to detect it continuously regardless of age.
  • the biological sample is preferably a biological sample from which the target nucleic acid has not been purified.
  • the biological sample of the present invention is treated by treatment other than purification of the target nucleic acid, for example, heating, cooling, centrifugation, enzyme treatment (treatment with an enzyme such as a glycolytic enzyme, lipolytic enzyme, or proteolytic enzyme).
  • a biological sample may be used.
  • cells or bacteria when cells or bacteria are contained in the biological sample, it can be treated with a surfactant in order to destroy the cells or bacteria. Thereby, for example, it is possible to destroy the cell membrane of cells contained in blood, the bacterial membrane of bacteria, and the like.
  • the reagent kit for detection or quantification of a target nucleic acid of the present invention is at least one compound selected from the group consisting of EGTA, BAPTA and derivatives thereof (hereinafter referred to as “the compound of the present invention” And an oligonucleotide for amplifying the target nucleic acid.
  • the compound of the present invention and the oligonucleotide for amplifying the target nucleic acid may be contained in one reagent, or may be contained in different reagents.
  • the reagent kit for detecting or quantifying a target nucleic acid of the present invention amplifies the target nucleic acid with a first reagent containing at least one compound selected from the group consisting of EGTA, BAPTA and derivatives thereof, or a salt thereof. And a second reagent containing an oligonucleotide for the purpose.
  • a reagent containing at least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof, or a salt thereof may be referred to as a “first reagent”, and for amplifying a target nucleic acid.
  • the reagent containing the oligonucleotides is sometimes referred to as “second reagent”.
  • a reagent comprising at least one compound selected from the group consisting of EGTA, BAPTA and derivatives thereof, or a salt thereof, and an oligonucleotide for amplifying a target nucleic acid is referred to as a “measuring reagent”.
  • Compound The compound used in the present invention is ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis (o-amino) Phenoxy) ethane-N, N, N ′, N′-tetraacetic acid (BAPTA) and their derivatives. These compounds are common in that their chemical structures have an ether bond and four carboxyl groups.
  • EGTA is a compound represented by the following chemical formula, and reacts with divalent and trivalent metal ions to form a complex.
  • a derivative of EGTA is a compound having a structure including an ether bond and four carboxyl groups, and a part of the chemical structure of EGTA (for example, a substituent) is substituted with another structure.
  • the number of ether bonds is preferably 1 to 3. Examples of such derivatives include 8-amino-2-[(2-amino-5-methylphenoxy) methyl] -6-methoxyquinoline-N, N, N ′, N′-tetraacetic acid.
  • BAPTA is a compound represented by the following chemical formula, and reacts with a divalent metal ion to form a complex.
  • a BAPTA derivative is a compound having a structure containing an ether bond and four carboxyl groups, wherein a part of the chemical structure of BAPTA (for example, a substituent) is substituted with another structure.
  • the number of ether bonds is preferably 1 to 3.
  • Examples of such derivatives include O, O′-bis (2-aminophenyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid tetraacetoxy ester, 1- [6-amino-2- ( 5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid, 1- [6-amino- 2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid pentaacetoxymethyl ester, 1- [2-Amino-5- (2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane
  • the compounds used in the present invention may form salts and hydrates.
  • the salt is not particularly limited as long as it has the effect of the present invention, and may form a salt with an acid or a salt with a base.
  • Examples of the salt with an acid include inorganic acids such as hydrochloride, hydrobromide, sulfate, and phosphate.
  • Examples of the salt with a base include alkali metal salts such as sodium salt and potassium salt, and chloride salts.
  • examples of the salt of the compound include 8-amino-2-[(2-amino-5-methylphenoxy) methyl] -6-methoxyquinoline-N, N, N ′, N′-tetraacetic acid tetra Potassium salt, 1,2-bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid tetrasodium salt, 1,2-bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid tetrapotassium salt, 1- [6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane -N, N, N ', N'-tetraacetic acid pentapotassium salt, 1- [2-amino-5- (6-carboxy-2-indolyl)
  • Examples of the hydrate of the compound of the present invention include 1,2-bis (o-aminophenoxy) ethane-N, N, N ′, N′-tetraacetic acid tetrapotassium hydrate.
  • a salt of a compound and a hydrate thereof may be represented only by the compound name.
  • a so-called free form which does not form a salt can be selected.
  • the above compounds and salts thereof are commercially available, and can be purchased from, for example, Dojindo Laboratories Co., Ltd.
  • the compound of the present invention has a function to suppress the nucleic acid amplification reaction inhibitor present in the test sample by mixing with the test sample containing the target nucleic acid. That is, in the present invention, the test sample can be used for the nucleic acid amplification reaction without purifying the nucleic acid from the test sample by the function of the compound of the present invention.
  • the concentration of the compound of the present invention is a mixture of a test sample containing a biological sample and a detection or quantification reagent containing the compound of the present invention and an oligonucleotide (hereinafter also referred to as “reaction mixture”).
  • the target nucleic acid contained in the test sample can be detected or quantified more rapidly and accurately when the concentration is within a certain range with respect to the concentration of the biological sample in the total amount. That is, in one embodiment of the present invention, at least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof, or a salt thereof is detected or detected including a test sample including a biological sample, the compound of the present invention, and an oligonucleotide.
  • the concentration of the compound of the present invention in the reaction mixture is, for example, 0.01 mM or more, 0.03 mM or more, 0.05 mM or more, 0.09 mM or more, 0.1 mM or more, 0.15 mM or more, 0.2 mM or more, 0.3 mM or more, 0.45 mM.
  • the concentration is 0.2 mM or more, or 0.3 mM or more, but is not limited thereto.
  • the upper limit of the concentration of the compound of the present invention in the total amount of the reaction mixture is not limited, and examples include a concentration of 10 mM or less.
  • an oligonucleotide can be arbitrarily determined, and those skilled in the art can detect or contain a test sample, a compound of the present invention, etc. and an oligonucleotide.
  • the amount or concentration of the compound of the present invention contained in the detection or quantification reagent is determined according to each volume of the reagent for quantification and the total amount of these mixtures, and the concentration of the compound of the present invention in the total amount of the mixture Can be produced so that the concentration is as exemplified above.
  • the test sample is 2 ⁇ l
  • the reagent containing the compound of the present invention is 2 ⁇ l
  • the oligonucleotide containing reagent is 16 ⁇ l
  • the total amount of these mixtures is 20 ⁇ l.
  • the reagent containing the compound of the present invention contains the compound of the present invention in the reagent containing the compound of the present invention at a concentration of 2 mM or more, whereby the reagent of the present invention in the total amount of the mixture
  • the compound of the present invention can be contained so that the concentration of the compound or the like is 0.2 mM or more.
  • the test sample when a biological sample is used as it is as a test sample without dilution, the test sample is 4 ⁇ l, and the detection or quantification reagent containing the compound of the present invention and the oligonucleotide is 16 ⁇ l.
  • the reagent containing the compound of the present invention contains the compound of the present invention in the detection or quantification reagent at a concentration of 0.75 mM or more, whereby the compound of the present invention in the total amount of the mixture
  • the compound of the present invention and the like can be contained so that the concentration thereof becomes 0.6 mM or more.
  • the test sample obtained by diluting a biological sample 5 times
  • the test sample is 10 ⁇ l (biological sample 2 ⁇ l + diluted solution 8 ⁇ l)
  • the reagent containing the compound of the present invention is 4 ⁇ l
  • the oligonucleotide is used.
  • the reagent containing 6 ⁇ l and the total amount of these mixtures is 20 ⁇ l
  • the reagent containing the compound of the present invention contains the compound of the present invention at a concentration of 1.5 mM or more, so that the present invention in the total amount of the mixture
  • the compound of the present invention can be contained so that the concentration of the compound or the like becomes 0.3 mM or more.
  • At least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof or a salt thereof is a test sample containing a biological sample, a compound of the present invention, etc.
  • the concentration in the total amount of the mixture is included in the reagent for detection or quantification so that the concentration is not less than the concentration calculated by the following formula A, and more preferably, the following formula B It is contained in the reagent for detection or quantification so as to be equal to or higher than the concentration calculated by
  • the upper limit of the concentration of at least one compound selected from the group consisting of EGTA, BAPTA and derivatives thereof in the total amount of the mixture or a salt thereof is not limited, but examples include a concentration of 10 mM or less. .
  • Formula A ⁇ [Biological sample in test sample (volume)] / [Total amount (volume) of mixture of test sample and detection or quantification reagent] ⁇ ⁇ 2 (mM)
  • Formula B ⁇ [Biological sample in test sample (volume)] / [Total amount (volume) of mixture of test sample and detection or quantification reagent] ⁇ ⁇ 3 (mM)
  • the test sample is 2 ⁇ l
  • the reagent containing the compound of the present invention is 2 ⁇ l
  • the oligonucleotide containing reagent is 16 ⁇ l
  • the total amount of these mixtures is 20 ⁇ l
  • the calculation using Equation A is as follows.
  • the test sample when a biological sample is used as it is as a test sample without dilution, the test sample is 4 ⁇ l, and the detection or quantification reagent containing the compound of the present invention and the oligonucleotide is 16 ⁇ l.
  • the total amount is 20 ⁇ l, the calculation using Equation B is as follows.
  • the compound of the present invention contains the test sample containing the biological sample and the detection or quantification reagent containing the compound of the present invention and the oligonucleotide in the total amount of the mixture. It is contained in the reagent for detection or quantification so that the concentration is 0.6 mM or more.
  • test sample obtained by diluting a biological sample 5 times
  • the test sample is 10 ⁇ l (biological sample 2 ⁇ l + diluted solution 8 ⁇ l)
  • the reagent containing the compound of the present invention is 4 ⁇ l
  • the oligonucleotide When the reagent is 6 ⁇ l and the total amount of these mixtures is 20 ⁇ l, the calculation using Equation B is as follows.
  • those skilled in the art can determine the concentration of the compound of the present invention in the total amount of the mixture by using the above formula A or B. Moreover, if the said density
  • the compound or the like of the present invention or a hydrate thereof can be used as it is, or it can be dissolved in a solvent and used in the form of a solution.
  • a solvent examples include water and a buffer solution.
  • buffer solution There are no limitations on the type of buffer, general acetate buffer, phosphate buffer, Tris-HCl buffer, phosphate-citrate buffer, MES, ADA, PIPES, ACES, BES, MOPS, TES HEPES, Tricine, Bicine, TAPS, CHES, CAPS, and the like can be used.
  • the pH of the buffer can be appropriately set by those skilled in the art.
  • the pH is 6.0 to 10.0, preferably 7.0 to 9.0, and more preferably 8.0 to 8.5.
  • the oligonucleotide used in the present invention is not limited as long as it contributes to amplification of the target nucleic acid, and examples thereof include oligonucleotide primers.
  • the oligonucleotide can be easily designed and synthesized by those skilled in the art depending on the base sequence of the target nucleic acid to be detected or quantified and the nucleic acid amplification method used.
  • base sequence information of a microorganism from which the target nucleic acid is derived is obtained from known gene information and gene databases (for example, NCBI GenBank, etc.), and a region specific to the microorganism (for example, a conserved region in the case of a virus, a toxin)
  • a base sequence of a toxin protein-encoding region is selected, and a nucleic acid having the base sequence is selected as a target nucleic acid.
  • Primer express manufactured by Applied Biosystems, Inc. so that all or part of the target nucleic acid can be amplified by any nucleic acid amplification method. It is possible to design by using production software such as The primer has, for example, a length of at least 10 bases, preferably at least 15 bases, more preferably at least 20 bases.
  • oligonucleotides specifically designed and commercialized for amplifying or detecting or quantifying a specific target nucleic acid can also be used.
  • DNA containing the human cytomegalovirus glycoprotein H gene can be selected as a target nucleic acid, and primers can be designed so that all or part of the target nucleic acid can be amplified.
  • primers can be designed so that all or part of the target nucleic acid can be amplified.
  • the primer used for nucleic acid amplification reaction can be used with the combination (primer set) of a some primer.
  • the number of primers included in the primer set is not limited, and can be set as appropriate according to the type of nucleic acid amplification method described below.
  • the target nucleic acid can be amplified by any nucleic acid amplification method.
  • the nucleic acid amplification method that can be used in the present invention include a method using a reaction involving temperature change (temperature cycle) and a method using an isothermal nucleic acid amplification reaction (hereinafter also referred to as “isothermal nucleic acid amplification method”). It is done.
  • the target nucleic acid is preferably amplified by an isothermal nucleic acid amplification method.
  • PCR method White, TJ et al., Trends Genet., 5, 185 (1989)
  • RT-PCR method LCR method (Ligase Chain Reaction: Barany, F., Proc. Natl. Acad. Sci. USA, Vol. 88, p.189-193, 1991).
  • SmartAmp Smart Amplification Process
  • LAMP Loop-Mediated Isothermal Amplification
  • SDA Strand Displacement Amplification: Edward L) Chan et al, Arch. Pathol. Lab.
  • amplification of the target nucleic acid is performed by PCR, RT-PCR, LCR, SmartAmp, LAMP, RT-LAMP, SDA, ICAN, TMA, NASBA, RCA, TRC.
  • the “SmartAmp method” includes a SmartAmp2 (Smart Amplification Process 2) method.
  • PCR method RT-PCR method, LCR method, SmartAmp method, LAMP method, SDA method, ICAN method, TMA method, NASBA method, RCA method, TRC method and HDA method are techniques well known to those skilled in the art. If it exists, it can implement easily based on well-known information.
  • the PCR method is a nucleic acid amplification reaction using PCR (polymerase chain reaction), and can amplify a large portion of a DNA portion sandwiched between two primers in a test tube.
  • the PCR method is a method of amplifying a nucleic acid by a reaction cycle of heat denaturation of DNA, primer annealing, and extension reaction with DNA polymerase, and involves a temperature change (temperature cycle).
  • RT-PCR is a method of synthesizing cDNA from RNA extracted from a biological sample by reverse transcription and amplifying a nucleic acid by PCR using the cDNA as a template.
  • DNA fragments amplified by PCR are visualized and quantified by labeling with fluorescent substances such as ethidium bromide and SYBER (registered trademark) GREENI and II that specifically bind to DNA after agarose gel electrophoresis, for example. be able to.
  • fluorescent substances such as ethidium bromide and SYBER (registered trademark) GREENI and II that specifically bind to DNA after agarose gel electrophoresis, for example. be able to.
  • the SmartAmp method is a nucleic acid amplification method using an isothermal nucleic acid amplification reaction, and is a method using 2 to 6 primer sets and a strand-displacement type DNA polymerase.
  • This method is a method of using a combination of a primer capable of forming a stem-loop only when a target nucleic acid is amplified and a primer having a folded sequence at the 5 ′ end in a nucleic acid amplification method using a strand displacement reaction. It is.
  • a special apparatus such as a thermal cycler is not required, and time required for temperature change is not required.
  • An amplification product can be obtained in a short time, and nucleic acid amplification can be performed more rapidly and with higher accuracy than the PCR method.
  • the primer nucleic acid can be denatured into a single strand to obtain a primer for the template nucleic acid. Annealing can also be performed efficiently. Examples of such a modification method include a method of raising the temperature to about 95 to 98 ° C. As another method, it is possible to denature by raising the pH, but in this case, it is necessary to lower the pH in order to hybridize the primer to the target nucleic acid.
  • Any strand-displacing DNA polymerase that can be used in the SmartAmp method may have any strand-displacement activity (strand displacement ability), and any of room temperature, intermediate temperature, and heat resistance may be used. can do. Further, this polymerase may be either a natural body or a mutant with artificial mutation. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that the DNA polymerase has substantially no 5 ' ⁇ 3' exonuclease activity.
  • Examples of such a DNA polymerase include Aac DNA polymerase having a strand displacement activity derived from a moderate thermoacidophilic bacterium Alicyclobacillus acidocaldarius (Aac), Bst DNA polymerase derived from Bacillus stearothermophilus, Bacillus cardio Examples include Bca DNA polymerase derived from Tenac (Bacillus caldotenax), Csa DNA polymerase, Klenow fragment of E. coli DNA polymerase I, etc., but from the group consisting of Aac DNA polymerase, Bst DNA polymerase and Csa DNA polymerase At least one selected DNA polymerase is preferred, and Aac DNA polymerase is more preferred.
  • the nucleic acid amplification reaction using the above primer set can be performed isothermally. Therefore, this nucleic acid amplification reaction includes a step of incubating a mixture of the test sample, the first reagent and the second reagent in the present invention isothermally.
  • “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function.
  • the “substantially constant temperature condition” means that not only the set temperature is accurately maintained but also a temperature change that does not impair the substantial functions of the enzyme and the primer is allowed. To do.
  • the nucleic acid amplification reaction under a constant temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained.
  • the reaction temperature is preferably set to a temperature near the melting temperature (Tm) of the primer or lower, and further, It is preferable to set the stringency level in consideration of the melting temperature (Tm) of the primer. Accordingly, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C, and even more preferably about 60 ° C to about 65 ° C.
  • nucleic acid amplification mechanism of the SmartAmp method Details such as the nucleic acid amplification mechanism of the SmartAmp method are described in International Publication WO 2005/063977 (Patent No. 3897805) and International Publication WO 2004/040019 (Patent No. 3867926), etc.
  • a person skilled in the art can design a primer based on these documents and the description in the present specification, and perform the SmartAmp method.
  • Reagents for amplifying nucleic acids by the SmartAmp method can be obtained from Danaform Co., Ltd.
  • the LAMP method anneals its 3 'end to the template nucleotide and uses it as a starting point for complementary strand synthesis. By combining primers that anneal to the loop formed at this time, isothermal complementary strand synthesis reaction is performed. This is a possible nucleic acid amplification method. In the LAMP method, using an inner primer pair or an outer primer pair, further adding a loop primer pair, two, four or six specific primers, a strand displacement polymerase and a substrate nucleotide, Amplify the target nucleic acid under isothermal conditions.
  • sequences complementary to each other are generated at the same strand upper end of the amplified product, and these are annealed to form a hairpin-like loop, and an elongation reaction by the polymerase starting from the loop occurs.
  • a strand displacement type extension reaction occurs from the primer annealed in the loop, and the extension product is dissociated into single strands. Since the dissociated single strand also has a complementary sequence at the end, this reaction occurs repeatedly.
  • the extension reaction and the amplification reaction proceed simultaneously at multiple positions on the same strand of the amplified product, DNA amplification is achieved exponentially and under isothermal conditions, and a small amount of nucleic acid is efficiently used. Can be amplified.
  • Real-time PCR devices include real-time PCR devices such as Mx3000p QPCR System (Agilent Technology Co., Ltd.) and ABIPRISM7500 (Applied Biosystems Co., Ltd.), which detect and monitor amplification products in real time to detect or quantify target nucleic acids. Is possible.
  • the amplified DNA can be detected with a fluorescent substance such as SYBER GREEN I described above.
  • a fluorescent probe can be used in combination with a primer.
  • a threshold is set for a certain fluorescence intensity (fluorescence) to be detected, and the target nucleic acid with a known concentration is amplified when the detected fluorescence intensity reaches the threshold when the test sample is amplified.
  • the target nucleic acid contained in the test sample can be quantified (or measured) more accurately by comparing with the time when the detected fluorescence intensity reaches the threshold value.
  • the threshold value is not limited as long as it is a value set so that the samples can be compared with each other.
  • the threshold value can be set within the range of the fluorescence intensity in the logarithmic amplification phase of the nucleic acid amplification curve. Such a threshold can be easily set by those skilled in the art.
  • the threshold value is set to a value that allows the fluorescence intensity associated with the amplification reaction to be sufficiently discriminated compared to the fluorescence intensity before amplification.
  • a test sample whose fluorescence intensity reaches a threshold value within a time period can be determined as positive, and a test sample whose fluorescence intensity does not reach a threshold value within a certain time can be determined as negative.
  • the reagent for detecting or quantifying the target nucleic acid of the present invention may contain a reagent for amplifying and / or detecting or quantifying the nucleic acid in addition to the oligonucleotide for amplifying the target nucleic acid.
  • a reagent for amplifying and / or detecting or quantifying the nucleic acid in addition to the oligonucleotide for amplifying the target nucleic acid.
  • examples of such a reagent include, but are not limited to, a nucleic acid amplification reaction mixture, a coloring reagent, and a DNA polymerase.
  • Examples of the mixture for nucleic acid amplification reaction include, but are not limited to, a substrate such as dNTPs, a buffer solution such as Tris-HCl buffer, tricine buffer, sodium phosphate buffer, potassium phosphate buffer, magnesium chloride (MgCl 2 ), A catalyst such as potassium chloride (KCl), ammonium sulfate (NH 4 ) 2 SO 4 and magnesium sulfate (MgSO 4 ), and a surfactant (such as Tween 20).
  • a fluorescent probe can be included.
  • a melting temperature adjusting agent can be added to the amplification reaction mixture in order to increase the amplification efficiency of the nucleic acid.
  • the melting temperature adjusting agent used in the present invention and its concentration in the reaction solution are appropriately selected by those skilled in the art in consideration of other reaction conditions that affect the hybridization conditions, such as salt concentration, reaction temperature, etc.
  • the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof, and more preferably dimethyl sulfoxide (DMSO) or betaine.
  • the coloring reagent is a reagent that emits fluorescence by binding to a nucleic acid, and can be used instead of a fluorescent probe.
  • the coloring reagent used in the present invention include, but are not limited to, a reagent that emits fluorescence when bound to double-stranded DNA, such as SYBR (registered trademark) GreenI.
  • SYBR registered trademark
  • GreenI Real-time PCR (quantitative PCR, qPCR) using SYBR (registered trademark) Green I does not require the preparation of a special fluorescently labeled probe for each target nucleic acid, and is inexpensive in terms of cost. it can.
  • DNA polymerases include Aac DNA polymerase, Bca DNA polymerase (BcaBEST DNA polymerase, Bca (exo-) DNA polymerase, etc.), Bst DNA polymerase, Csa DNA polymerase, Klenow fragment, Vent DNA polymerase, Vent DNA (Exo -) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, ⁇ 29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9 °
  • Examples include Nm DNA polymerase, Therminater DNA polymerase, and the like, but at least one DNA DNA polymerase selected from the group consisting of Aac DNA polymerase, Bst DNA polymerase, and Csa DNA polymerase is preferable.
  • the type of DNA polymerase can be appropriately selected by those skilled in the art according to the nucleic acid amplification method used. For example, when the SmartAmp method is used, Aac DNA polymerase can be selected, and when the LAMP method is used, Bst DNA polymerase or Csa DNA polymerase can be selected, but is not limited thereto.
  • an enzyme stabilizer may be added to the target nucleic acid detection or quantification reagent of the present invention.
  • the enzyme stabilizer used in the present invention may be any known in the art, such as glycerol, bovine serum albumin, and saccharides, and is not particularly limited.
  • a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can be added to the reaction solution as an enzyme stabilizer. .
  • Such a reagent may be any known in the art and is not particularly limited, but is preferably a saccharide, more preferably a mono- or oligosaccharide, more preferably trehalose, sorbitol or mannitol, or these The mixture of 2 or more types of these is mentioned.
  • the reagent kit for detecting or quantifying the target nucleic acid of the present invention may be any kit as long as it contains at least a reagent containing the compound of the present invention and an oligonucleotide, and may contain other components. Examples of other components include a reaction container (such as a test tube) and an instruction manual. Also, some of the components of the detection or quantification reagent can be included in the kit as separate reagents.
  • “detection” of a target nucleic acid means confirmation of amplification of the target nucleic acid.
  • the target nucleic acid contained in the test sample when a nucleic acid amplification reaction is performed, if the amount of amplification of the target nucleic acid contained in the test sample is higher than that of a control not containing the target nucleic acid, it can be said that the target nucleic acid contained in the test sample has been detected. In a more preferred embodiment, it can be said that the target nucleic acid has been detected when the amplification amount of the target nucleic acid contained in the test sample reaches a certain value within a certain time.
  • “quantification” of the target nucleic acid means measuring the amount (for example, copy number) of the target nucleic acid contained in the test sample.
  • a target nucleic acid derived from a virus or bacteria can be amplified and detected or quantified. Therefore, for example, by detecting or quantifying a target nucleic acid derived from a virus or bacteria contained in a biological sample collected from an animal, it is possible to detect or diagnose viral infection or bacterial infection. That is, the reagent kit for detecting or quantifying the target nucleic acid of the present invention can be used for detecting or diagnosing viral infection or bacterial infection, and thus can be used as a diagnostic kit for viral infection or bacterial infection.
  • the detection or quantification kit of the present invention detection or quantification of a herpes virus-derived target nucleic acid contained in urine collected from a human patient, the presence or absence of herpes virus infection in the patient Diagnosis can be made. More specifically, it is possible to diagnose congenital cytomegalovirus infection by detecting or quantifying the target nucleic acid derived from human cytomegalovirus contained in urine collected from a newborn within 3 weeks of birth. In addition, by detecting or quantifying human cytomegalovirus-derived target nucleic acid contained in urine collected from infants, it is possible to diagnose the presence or absence of infection including acquired cytomegalovirus infection. . As a target nucleic acid derived from human cytomegalovirus, DNA containing the base sequence of the glycoprotein H gene of human cytomegalovirus can be selected.
  • the present invention relates to ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis (o -Aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA) and at least one compound selected from the group consisting of these derivatives, or a salt thereof, a test sample, and the target nucleic acid is amplified. And a method for detecting or quantifying the amplified target nucleic acid by mixing with an oligonucleotide for the purpose, performing an amplification reaction of the target nucleic acid in the mixture.
  • EGTA ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid
  • BAPTA 1,2-bis (o -Aminophenoxy) ethane-N, N, N ', N'-
  • the present invention also relates to (a) ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane-
  • EGTA ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid
  • BAPTA 1,2-bis (o-aminophenoxy) ethane-
  • a first reagent containing at least one compound selected from the group consisting of N, N, N ′, N′-tetraacetic acid (BAPTA) and derivatives thereof, or a salt thereof, a test sample, and a target nucleic acid A step of mixing with a second reagent containing the oligonucleotide of (b), and (b) a step of performing an amplification reaction of the target nucleic acid in the mixture obtained in step (a)
  • the step (a) may be a step of mixing the first reagent and the test sample, and mixing all or a part of the mixture with the second reagent, or the first reagent and the second sample.
  • the step may be a step of mixing all or a part of the mixture with the test sample.
  • the process of mixing a test sample and a 2nd reagent and mixing all or one part of the mixture and a 1st reagent may be sufficient.
  • the mixture of the first reagent and the second reagent that is, the reagent containing the compound of the present invention and the oligonucleotide for amplifying the target nucleic acid
  • a test sample is mixed with a reagent containing at least one compound selected from the group consisting of EGTA, BAPTA, and derivatives thereof, or a salt thereof, and an oligonucleotide for amplifying a target nucleic acid. It may be a process.
  • the description of the first reagent, the test sample, and the second reagent is as described above.
  • mixing can be performed by a commonly used method. Examples of such a method include, but are not limited to, pipetting, tapping, and stirring with a stirrer (Vortex mixer or the like).
  • “all” of the mixture means the total amount of the mixture.
  • the second reagent when the second reagent is added to the mixture of the first reagent and the test sample, the entire mixture of the first reagent and the test sample and the second reagent are mixed.
  • the “part” of the mixture means a solution of a part of the mixture.
  • a part is taken from the mixture of the first reagent and the test sample, and this is used as the second reagent.
  • Step (b) is a step of amplifying a target nucleic acid in a mixture (reaction mixture) of a first reagent, a test sample, and a second reagent by a nucleic acid amplification reaction, and detecting or quantifying the amplified target nucleic acid.
  • the description of the nucleic acid amplification method that can be used in the present invention is as described above. In the nucleic acid amplification reaction, the entire reaction mixture can be used, or a part of the solution can be used.
  • the target nucleic acid amplified by the nucleic acid amplification reaction can be detected or quantified by any method.
  • Examples of such a method include a real-time PCR apparatus, general gel electrophoresis, a method using a labeled probe, Northern blotting, Southern blotting, dot blot, and a method using immunochromatography.
  • the method using is preferable.
  • Detection or quantification using a real-time PCR apparatus can be performed using a real-time PCR apparatus such as Mx3000p QPCR System (Agilent Technology Co., Ltd.) or ABIPRISM7500 (Applied Biosystems Co., Ltd.).
  • a fluorescent substance such as ethidium bromide or SYBR (registered trademark) GreenI.
  • Examples of the method using a labeled probe include a method of detecting by using a labeled probe having a label such as biotin and hybridizing it to an amplification product.
  • viral infection or bacterial infection can be detected by selecting a target nucleic acid derived from a virus or bacteria as the target nucleic acid. That is, the present invention relates to ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane-N, N , N ′, N′-tetraacetic acid (BAPTA) and derivatives thereof, at least one compound or a salt thereof, a test sample, and an oligonucleotide for amplifying a target nucleic acid derived from a virus or a bacterium
  • a method of detecting a viral infection or a bacterial infection comprising the steps of performing amplification reaction of the target nucleic acid in the mixture, and detecting or quantifying the amplified target nucleic acid.
  • this invention provides the method of detecting viral infection or bacterial infection including the following processes.
  • a step of mixing with a second reagent containing an oligonucleotide and (b) a step of carrying out an amplification reaction of the target nucleic acid in the mixture obtained in step (a) and detecting or quantifying the amplified target nucleic acid.
  • the explanation regarding the steps (a) and (b) is the same as the method for detecting or quantifying
  • a threshold is set for a certain fluorescence intensity (Fluorescence) to be detected, and the time when the detected fluorescence intensity when the test sample is amplified reaches the threshold within a specified time Can be quantified (or measured) the target nucleic acid contained in the test sample by comparing with the time when the detected fluorescence intensity when the target nucleic acid of known concentration is amplified reaches the threshold value, and thus more accurately the virus infection or Bacterial infection can be detected.
  • the threshold value is not limited as long as it is a value set so that the samples can be compared with each other.
  • a value that allows the fluorescence intensity associated with the amplification reaction to be sufficiently distinguished from the fluorescence intensity before amplification is set as a threshold, and within a certain time
  • the test sample whose fluorescence intensity has reached the threshold value can be detected or diagnosed as positive, and the test sample whose fluorescence intensity does not reach the threshold value within a certain time can be determined or diagnosed as negative. That is, the method of the present invention provides a diagnostic method in addition to a method for detecting viral infection or bacterial infection.
  • the fixed time from the start of the nucleic acid amplification reaction to the detection is not limited, but is, for example, about 30 to 90 minutes, preferably about 30 to 60 minutes, more preferably about 40 to 60 minutes.
  • the first reagent is ethylene glycol-bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (EGTA (GEDTA)) and 1,2-bis (o-amino) Phenoxy) ethane-N, N, N ′, N′-tetraacetic acid (BAPTA) (Dojindo Laboratories) was dissolved in a 10 mM Tris-HCl (pH 8.0) solution.
  • EGTA ethylene glycol-bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid
  • BAPTA 1,2-bis (o-amino) Phenoxy) ethane-N, N, N ′, N′-tetraacetic acid
  • Second Reagent The second reagent was prepared so that real-time PCR can be performed using various nucleic acid amplification methods.
  • a reagent for carrying out the SmartAmp method SmartAmp method reagent
  • a reagent for carrying out the LAMP method LAMP method reagent
  • the composition and production method of each reagent are shown below.
  • the concentration in the composition of each reagent indicates the final concentration relative to the total amount of the mixture of the test sample, the first reagent, and the second reagent (reaction mixture).
  • Primer mixture The primer mixture was prepared by mixing the following primers (100 ⁇ M) of TP, FP, BP, OP1 and OP2 at a ratio of 8: 8: 4: 1: 1.
  • TP 5'- CGCGTCTCTCCAAGAACTCTACCTCATG SEQ ID NO: 4
  • FP 5'- TTTATATATATATAAACGTTTCTACGATGAAGAT SEQ ID NO: 5
  • BP 5'- CATGGAGTGGACGA SEQ ID NO: 6
  • OP1 5'-GCCGACTTTGCCCTA SEQ ID NO: 7
  • OP2 5'- CAACTGCGTAAAGTGTG SEQ ID NO: 8
  • Primer mixture The primer mixture is prepared by mixing the following FIP, BIP, LPF, LPB, F3 and B3 primers (100 ⁇ M) in a ratio of 8: 8: 4: 4: 1: 1. Produced.
  • the nucleotide sequence of each primer was according to Table 1 of Eiko Fukushima, et al., Journal of Virological Methods 151 (2008) 55-60.
  • FIP F1c-F2 5'- gatttcgcgtctctccgtcgt-gaactctacctcatgggcag (SEQ ID NO: 9)
  • BIP B1-B2c 5'- catcgtagaaacgggcctctg-tgcggatgagctagcaactg (SEQ ID NO: 10)
  • LPF LPF
  • LPB LPB
  • F3 F3
  • B3c 5'-gggtctcttttcttcagcct (SEQ ID NO: 13)
  • HCMV solution was prepared as follows.
  • the HCMV towne strain was obtained from ATCC (American Type Culture Collection) and inoculated into a human fetal lung-derived normal fibroblast cell line (HFL-III).
  • the inoculated cell line was cultured in a medium in which FBS was added to D-MEM / Ham's F-12 with L-Glutamine and Phenol Red (Wako) and grown by a general method.
  • the culture solution after the proliferation of the cells was centrifuged, and the supernatant collected after centrifugation was used as an HCMV solution (5 ⁇ 10 6 pfu / mL).
  • test sample to the 0.2 mL PCR tube (Agilent Technologies) in the amount shown in Table 1 below (2, 3 or 4 ⁇ l), and display EGTA.
  • An additional 2 ⁇ l of the first reagent at each concentration shown in 2-4 was added and spun down.
  • heat treatment at 98 ° C. for 30 seconds was performed, followed by ice cooling.
  • the second reagent was added thereto, followed by spin-down, and then nucleic acid amplification reaction and target nucleic acid detection by SmartAmp method were performed using Mx3000p QPCR System (Agilent Technologies Inc.).
  • Table 1 shows the composition of a mixture (reaction mixture) of the test sample, the first reagent, and the second reagent.
  • the water volume is appropriately selected as 2, 3 or 4 ⁇ l depending on the amount of test sample added so that the volume of the mixture of the test sample, first reagent and second reagent is 20 ⁇ l. did.
  • Nucleic acid amplification reaction and target nucleic acid detection by the SmartAmp method were performed under the following conditions.
  • Reaction time Reaction at 60 ° C. for 60 minutes.
  • Measurement wavelength detected at excitation 492 nm, fluorescence 516 nm.
  • Judgment criteria for detection The amount of change in fluorescence intensity relative to the fluorescence intensity before amplification (hereinafter referred to as “Fluorescence (dR)”) is compared with the time (minutes) to reach 4000, and positive when it reaches 4000 within 60 minutes It was determined.
  • the fluorescence (dR) reaches 4000 within 60 minutes in both the specimens 7 and 8. Positive.
  • the nucleic acid was not purified from the test sample. It was shown that the target nucleic acid derived from HCMV contained in urine can be detected.
  • the first reagent containing EGTA so that the concentration of EGTA relative to the total amount of the reaction mixture is equal to or higher than the concentration calculated by the above formula A or B, detection is possible when a reagent not containing EGTA is used. It was shown that target nucleic acids derived from HCMV in urine that could not be detected can be detected.
  • Nucleic acid amplification reaction and target nucleic acid detection by the LAMP method were performed under the following conditions.
  • Reaction time Reaction at 60 ° C. for 45 minutes.
  • Measurement wavelength detected at excitation 492 nm, fluorescence 516 nm.
  • Detection criteria The time (minutes) at which Fluorescence (dR) reaches 1000 was compared, and the case where it reached 1000 within 45 minutes, which is a general determination time in the LAMP method, was detected.
  • the urine was purified without purifying the nucleic acid from the test sample. It was shown that the target nucleic acid derived from HCMV contained in can be detected. It was also shown that HCMV-derived target nucleic acid contained in urine can be rapidly detected within 45 minutes by using the first reagent containing EGTA so that the concentration of EGTA relative to the total amount of the reaction mixture is 0.45 ⁇ mM or higher. It was done.
  • the concentration of EGTA relative to the total amount of the reaction mixture is equal to or higher than the concentration calculated by the above formula A or B. It was shown that target nucleic acids derived from HCMV in urine that could not be detected can be detected.
  • HCMV human cytomegalovirus
  • saliva determined to be CMV-negative as a result of real-time PCR
  • the ratio of the HCMV solution contained in the test sample is 0.25%.
  • the negative saliva saliva not containing HCMV was collected from a healthy human in a 1.5 ml tube.
  • the HCMV solution was prepared in the same manner as described in Example 2.
  • Example 2 Detection of target nucleic acid in virus-positive saliva
  • Table 9 shows the composition of the mixture (reaction mixture) of the test sample, the first reagent, and the second reagent in this example. Of the following composition, 4 ⁇ l of water was added such that the volume of the mixture of the test sample, the first reagent and the second reagent was 20 ⁇ l. Table 9
  • Nucleic acid amplification reaction and target nucleic acid detection by the SmartAmp method were performed under the following conditions.
  • Reaction time Reaction at 60 ° C. for 60 minutes.
  • Measurement wavelength detected at excitation 492 nm, fluorescence 516 nm.
  • Detection criteria The time (minutes) for Fluorescence (dR) to reach 1000 was compared, and the case where it reached 1000 within 60 minutes was determined as positive.
  • saliva was obtained without purifying the nucleic acid from the test sample by using the first reagent containing EGTA and the second reagent containing the oligonucleotide for amplifying the target nucleic acid by the SmartAmp method. It was shown that the target nucleic acid derived from HCMV contained in can be detected. Further, it was shown that the target nucleic acid derived from HCMV contained in saliva can be detected by using the first reagent containing EGTA so that the concentration of EGTA with respect to the total amount of the reaction mixture is 1 ⁇ mM or higher.
  • CMV genome 5000copies / mL urine or more
  • positive urine 11 people from unpurified urine (hereinafter referred to as “CMV genome strongly positive urine”) that have been found to contain 10 6 copies / mL urine or more of CMV genome.
  • Positive urine hereinafter referred to as “strongly positive urine” added to each unpurified negative urine sample collected from humans at a rate of 10% was prepared (sample numbers S6 to S15).
  • the first reagent (EGTA- (0 mM)) containing no EGTA is used in any of sample numbers S6 to S15.
  • a remarkable difference was confirmed when the first reagent having an EGTA concentration of 10 mM (concentration with respect to the total amount of the reaction mixture was 1 mM) was used.
  • the Fluorescence (dR) did not reach 4000 within 60 minutes.
  • the concentration of EGTA is the same as that when using the first reagent (EGTA- (0 mM)) not containing EGTA in specimen numbers S2 to S5.
  • a significant difference was observed when the first reagent of 10 mM (concentration with respect to the total amount of the reaction mixture was 1 mM) was used.
  • Fluorescence (dR) did not reach 4000 within 60 minutes (that is, the detection result was negative).
  • the first reagent containing EGTA and the oligonucleotide containing the oligonucleotide for amplifying the target nucleic acid by the SmartAmp method are used. It was shown that by using the two reagents, the target nucleic acid derived from HCMV contained in urine can be detected without purifying the nucleic acid from the test sample. Moreover, it was shown that the target nucleic acid derived from HCMV in urine that could not be detected when using a reagent not containing EGTA can be detected by the present invention.
  • Detection of target nucleic acid in herpes simplex virus type 1 (HSV-1) positive urine In this example, detection of a target nucleic acid of herpes simplex virus type 1 (HSV-1) was performed. Further, the detection was performed using a single reagent containing EGTA and the LAMP method reagent without dividing the reagent used for detecting the target nucleic acid into the first reagent and the second reagent.
  • a reagent (hereinafter referred to as “measurement reagent”) containing EGTA and a LAMP method reagent was prepared.
  • the composition and production method of the measurement reagent are shown below.
  • the concentration in the composition of the measurement reagent indicates the final concentration with respect to the total amount of the mixture of the test sample and the measurement reagent (reaction mixture).
  • Primer mixture contains the following HSV1 FIP, HSV1 BIP, HSV1 LPF, HSV1 LPB, HSV1 F3 and HSV1 B3 primers (100 ⁇ M) at 8: 8: 4: 4: 1: 1 It was prepared by mixing at a ratio of The base sequence of each primer was according to Table 1 of AK Reddy et al., Clin. Microbiol Infect, 2011; 17: 210-213.
  • HSV1 FIP (F1-F2) 5'- ccagacgttccgttggtaggtcttttacttttgactgttcgcgcacc (SEQ ID NO: 15)
  • HSV1 BIP (B1-B2) 5'- ccatcatcgccacgtcggacttttttcggcgtctgcttttgtg (SEQ ID NO: 16)
  • HSV1 LPF 5'-aaatcctgtcgccctacacagcgg
  • HSV1 LPB 5'- caccccgcgacgggacgccg
  • HSV1 F3 5'- cagccacacacctgtgaa (SEQ ID NO: 19)
  • HSV1 B3 5'- tccgtcgaggcatcgttag (SEQ ID NO: 20)
  • Virus-positive urine of herpes simplex virus type 1 A sample (virus-positive urine) obtained by adding an HSV-1 solution to urine collected from a healthy human was used as a test sample. The percentage of HSV-1 solution contained in the test sample is 0.25%. Urine was collected in a urine collection pack or paper cup. HSV-1 was obtained from ABI (Advanced Biotechnologies Inc.).
  • Nucleic acid amplification reaction and target nucleic acid detection by the LAMP method were performed under the following conditions.
  • Reaction time Reaction at 65 ° C. for 45 minutes (Study 1 below).
  • Measurement wavelength detected at excitation 492 nm, fluorescence 516 nm.
  • Detection criteria The time (minutes) at which Fluorescence (dR) reaches 1000 was compared, and the case where it reached 1000 within 45 minutes, which is a general determination time in the LAMP method, was detected.
  • the target nucleic acid derived from HSV-1 contained in urine can be obtained without purifying the nucleic acid from the test sample. It was shown to be detectable. In addition, it was shown that HSV-1-derived target nucleic acid contained in urine can be rapidly detected within 45 minutes by using a measurement reagent containing EGTA so that the concentration of EGTA relative to the total amount of the reaction mixture is 0.2 ⁇ mM or higher. It was done.
  • virus infection or bacterial infection can be detected by the present invention. Furthermore, it was shown that the present invention can be used for diagnosis of viral infection or bacterial infection by judging positive and negative based on the time to reach the threshold value of detected fluorescence intensity (Threshold).
  • the present invention provides a novel nucleic acid detection reagent kit and detection method capable of detecting a target nucleic acid contained in a biological sample quickly and accurately without purifying the nucleic acid from the biological sample.

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Abstract

La présente invention porte sur un nouveau kit de réactifs et un nouveau procédé pour détecter ou doser un acide nucléique qui permettent de détecter ou de doser rapidement et à une précision élevée un acide nucléique cible contenu dans un échantillon biologique sans purification dudit acide nucléique à partir de l'échantillon biologique. Le kit de réactifs pour détecter ou doser un acide nucléique selon l'invention comprend au moins un composé choisi dans le groupe constitué par l'acide éthylène glycol-bis(2-aminoéthyléther)-N,N,N',N'-tétraacétique (EGTA), l'acide 1,2-bis(o-aminophénoxy)éthane-N,N,N',N'-tétraacétique (BAPTA) et leurs dérivés, et un oligonucléotide pour l'amplification de l'acide nucléique cible.
PCT/JP2013/059307 2012-03-29 2013-03-28 Procédé de détection ou de dosage d'acide nucléique et kit de réactifs afférent WO2013147043A1 (fr)

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JP2017501704A (ja) * 2013-12-24 2017-01-19 ゼネラル・エレクトリック・カンパニイ タンパク質の安定化及び保存のための静電紡糸繊維
CN109402272A (zh) * 2018-08-24 2019-03-01 暨南大学 基于智能恒温扩增技术检测单核增生李斯特氏菌的引物组、试剂盒及方法
JP2019517778A (ja) * 2016-04-08 2019-06-27 スリーエム イノベイティブ プロパティズ カンパニー 細胞溶解及び核酸増幅のためのプロセス

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JP2001526051A (ja) * 1997-12-10 2001-12-18 シエラ ダイアグノスティクス，インク． 体液中のｄｎａを保存するための方法及び試薬
JP2002199899A (ja) * 2000-11-15 2002-07-16 Becton Dickinson & Co 細胞と核酸標的の保存方法
JP2006246792A (ja) * 2005-03-10 2006-09-21 Bussan Nanotech Research Institute Inc 微生物検出方法及び溶菌試薬

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017501704A (ja) * 2013-12-24 2017-01-19 ゼネラル・エレクトリック・カンパニイ タンパク質の安定化及び保存のための静電紡糸繊維
JP2019517778A (ja) * 2016-04-08 2019-06-27 スリーエム イノベイティブ プロパティズ カンパニー 細胞溶解及び核酸増幅のためのプロセス
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CN109402272A (zh) * 2018-08-24 2019-03-01 暨南大学 基于智能恒温扩增技术检测单核增生李斯特氏菌的引物组、试剂盒及方法

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