WO2011074181A1 - Procédé de détection complète de cinq types de virus vih, vhc, vhb, pvb19 et wnv ayant chacun de multiples génotypes, ensembles d'amorces, puces à adn et nécessaire de détection des virus - Google Patents

Procédé de détection complète de cinq types de virus vih, vhc, vhb, pvb19 et wnv ayant chacun de multiples génotypes, ensembles d'amorces, puces à adn et nécessaire de détection des virus Download PDF

Info

Publication number
WO2011074181A1
WO2011074181A1 PCT/JP2010/006687 JP2010006687W WO2011074181A1 WO 2011074181 A1 WO2011074181 A1 WO 2011074181A1 JP 2010006687 W JP2010006687 W JP 2010006687W WO 2011074181 A1 WO2011074181 A1 WO 2011074181A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
seq
sequence represented
base sequence
primer
Prior art date
Application number
PCT/JP2010/006687
Other languages
English (en)
Japanese (ja)
Inventor
龍生 中島
功 浜口
和也 滝澤
哲也 水谷
大二 遠藤
Original Assignee
株式会社日本パーカーライジング広島工場
国立感染症研究所
学校法人酪農学園
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 株式会社日本パーカーライジング広島工場, 国立感染症研究所, 学校法人酪農学園 filed Critical 株式会社日本パーカーライジング広島工場
Publication of WO2011074181A1 publication Critical patent/WO2011074181A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB

Definitions

  • the present invention relates to a method for detecting a virus in a biological sample, a primer set for detecting a virus, a microarray, and a kit for detecting a virus.
  • HIV Human Immunodeficiency Virus
  • CD4-positive T cells play a central role in the body's immunity
  • a decrease in the number of CD4 cells means a decrease in immunity, which ultimately results in immunodeficiency and usually becomes infected. Infected with no pathogen (opportunistic infection), will develop or cause malignant lymphoma.
  • HCV Hepatitis® Virus
  • HCV Hepatitis® Virus
  • the HCV gene is a plus-strand RNA consisting of about 9600 bases.
  • Hepatitis B that develops by infection with HBV (Hepatitis B Virus) is a viral infectious disease like hepatitis C and causes dangerous diseases such as chronic hepatitis and cirrhosis. HBV is often infected via blood and body fluids.
  • HBV is a DNA virus consisting of a nucleocapsid with a diameter of 27 nm, which is a spherical particle with a diameter of 42 nm, enveloping a 7 nm envelope (envelope), circular double-stranded (partly single-stranded) DNA, DNA polymerase, reverse transcriptase and the like.
  • the HBV gene is a circular double-stranded DNA consisting of about 3200 bases.
  • PvB19 (Parvovirus B19) is a virus that causes parvovirus B19 infection and is a common childhood disease that usually has a minor course in individuals with normal immunity and is known as infectious erythema or fifth disease Rash occurs. However, the virus has been found to be associated with hepatitis and myocarditis, and when the mother is infected during pregnancy, the virus can be transmitted to the fetus, causing hydrosis, spontaneous abortion, or in utero death. cause.
  • the PvB19 gene is a linear DNA consisting of about 5500 bases.
  • WNV West Nile Virus
  • West Nile virus infection such as West Nile encephalitis or West Nile fever.
  • WNV maintains an infection ring between birds and mosquitoes and infects humans and horses mainly through mosquitoes.
  • the main clinical manifestations of West Nile virus infection in humans are fever, headache, back pain, muscle pain, weakness, loss of appetite, skin rash, and swelling of lymph nodes, and some patients develop encephalomyelitis.
  • West Nile virus is a spherical positive-strand RNA virus having a diameter of about 50 nm and an envelope.
  • viruses are listed as factors that cause extremely dangerous diseases, and early identification of the causative viruses is very important for establishing prevention, diagnosis, and treatment methods for diseases and diseases.
  • virus testing based on immunological measurement of antigens and antiviral antibodies, there are many cases in which antigen testing cannot be used for diagnosis because the amount of virus is below the measurement sensitivity, despite being in an infectious state.
  • PCR polymerase chain reaction
  • the present invention is a detection for comprehensively detecting five kinds of viruses of a plurality of genotypes in biological samples: HIV, HCV, HBV, PvB19 and WNV with high specificity and sensitivity in a single detection operation. It is an object of the present invention to provide a method, and a virus detection primer set, a microarray, and a virus detection kit used for detection.
  • the present inventors amplify five kinds of viral genes of HIV, HCV, HBV, PvB19 and WNV having a plurality of genotypes using specific primers by PCR, and PCR amplification Primer set capable of amplifying five types of viral genes having the above-mentioned plurality of genotypes as a result of intensive studies focusing on a gene detection method characterized by hybridizing and detecting a product with a microarray, High specificity and sensitivity in five types of viral genes of HIV, HCV, HBV, PvB19 and WNV having multiple genotypes by using probe sets and detection conditions capable of detecting five types of viral genes simultaneously Thus, it was found that the detection can be performed quickly and easily, and the present invention has been completed.
  • the present invention is a method for detecting a pathogenic virus, (1) Primer set 1 composed of a primer consisting of the base sequence shown in SEQ ID NO: 1 and a primer consisting of the base sequence shown in SEQ ID NO: 2 for amplifying the human immunodeficiency virus (HIV) gene, (2) Primer set 2 composed of a primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 4 for amplifying the hepatitis C virus (HCV) gene; (3) Primer set 3 composed of a primer consisting of the base sequence shown in SEQ ID NO: 5 and a primer consisting of the base sequence shown in SEQ ID NO: 6 for amplifying the hepatitis B virus (HBV) gene; (4) Primer set 4 composed of a primer consisting of the base sequence shown by SEQ ID NO: 7 and a primer consisting of the base sequence shown by SEQ ID NO: 8 for amplifying the parvovirus B19 (Pv
  • the (1) HIV gene is one or two or more oligonucleotides comprising the nucleotide sequence represented by SEQ ID NO: 11 (Genbank Accession No. NC_001802 from 4153th to 4464th) or a complementary nucleotide sequence thereof
  • the (2) HCV gene is a nucleotide sequence represented by SEQ ID NO: 12 (59th to 327th of GenBank Accession No.
  • the (3) HBV gene includes one or two or more oligonucleotides comprising the nucleotide sequence represented by SEQ ID NO: 13 (from 450 to 715 of GenBank Accession No. X — 70185) or its complementary nucleotide sequence,
  • the (4) PvB19 gene is a nucleotide sequence represented by SEQ ID NO: 14 (from No. 2143 to No. 2278 of GenBank Accession No.
  • the (5) WNV gene is one or more oligonucleotides comprising the nucleotide sequence represented by SEQ ID NO: 15 (GenBank Accession No. NC — 009942, 3445th to 3599th) or its complementary nucleotide sequence It is characterized by that.
  • the pathogenic virus detection method of the present invention is characterized in that the one or more viral genes are amplified by PCR.
  • the present invention is a method for detecting a pathogenic virus,
  • a probe comprising the base sequence represented by SEQ ID NO: 16 (probe 1), A probe (probe 2) comprising the base sequence represented by SEQ ID NO: 17, A probe comprising the base sequence represented by SEQ ID NO: 18 (probe 3), A probe comprising the nucleotide sequence represented by SEQ ID NO: 19 (probe 4), A probe comprising the base sequence represented by SEQ ID NO: 20 (probe 5), A probe comprising the nucleotide sequence represented by SEQ ID NO: 21 (probe 6), A probe comprising the base sequence represented by SEQ ID NO: 22 (probe 7)
  • a probe set 1 comprising:
  • a probe comprising the base sequence represented by SEQ ID NO: 23 (probe 8), A probe (probe 9) comprising the base sequence represented by SEQ ID NO: 24, A probe comprising the base sequence represented by SEQ ID NO:
  • one or more probe sets selected from the group consisting of the probe sets 1 to 5 are fixed on a microarray substrate.
  • a primer set 1 comprising a primer consisting of a base sequence represented by SEQ ID NO: 1 and a primer consisting of a base sequence represented by SEQ ID NO: 2 for amplifying the human immunodeficiency virus (HIV) gene
  • Primer set 2 comprising a primer consisting of a base sequence represented by SEQ ID NO: 3 and a primer consisting of a base sequence represented by SEQ ID NO: 4 for amplifying the hepatitis C virus (HCV) gene
  • a primer set 3 comprising a primer consisting of a base sequence represented by SEQ ID NO: 5 and a primer consisting of a base sequence represented by SEQ ID NO: 6 for amplifying the hepatitis B virus (HBV) gene
  • Primer set 4 composed of a primer consisting of the base sequence shown by SEQ ID NO: 7 and a primer consisting of the base sequence shown by SEQ ID NO: 8 for amplifying the parvovirus B19 (PvB19) gene
  • the primer is fluorescently labeled, and the fluorescent label is made of Cy3 or Cy5.
  • a probe comprising the base sequence represented by SEQ ID NO: 16 (probe 1), A probe (probe 2) comprising the base sequence represented by SEQ ID NO: 17, A probe comprising the base sequence represented by SEQ ID NO: 18 (probe 3), A probe comprising the nucleotide sequence represented by SEQ ID NO: 19 (probe 4), A probe comprising the base sequence represented by SEQ ID NO: 20 (probe 5), A probe comprising the nucleotide sequence represented by SEQ ID NO: 21 (probe 6), A probe comprising the base sequence represented by SEQ ID NO: 22 (probe 7)
  • a probe set 1 comprising:
  • a microarray characterized in that one or more probe sets selected from the group consisting of the probe sets 1 to 5 are fixed on a microarray substrate.
  • a virus detection kit comprising any one of the above-described primer, probe set, and microarray.
  • the present invention can detect five types of viruses, such as HIV, HCV, HBV, PvB19 and WNV, having a plurality of genotypes in a biological sample, for example, blood transfused with a single detection operation with high specificity and sensitivity.
  • An exhaustive detection method, a virus detection primer set, a microarray, and a virus detection kit are provided. It becomes possible to inspect and analyze various types of viruses more quickly than conventional techniques.
  • FIG. 1 is a flowchart illustrating primer design for target sequences of each viral gene.
  • FIG. 2 is an explanatory diagram of agarose gel electrophoresis in which it was confirmed that a primer can specifically amplify a viral gene for each virus subtype gene.
  • FIG. 3 is an explanatory diagram confirming the specificity between the probe candidates shown in Table 3 and the target sequence of the HIV gene.
  • FIG. 4 is an explanatory diagram confirming the specificity between the probe candidates shown in Table 3 and the target sequence of the HCV gene.
  • FIG. 5 is an explanatory diagram confirming the specificity between the probe candidates shown in Table 3 and the target sequence of the HBV gene.
  • FIG. 1 is a flowchart illustrating primer design for target sequences of each viral gene.
  • FIG. 2 is an explanatory diagram of agarose gel electrophoresis in which it was confirmed that a primer can specifically amplify a viral gene for each virus subtype gene.
  • FIG. 3 is an explanatory diagram confirming
  • FIG. 6 is an explanatory diagram confirming the specificity between the probe candidates shown in Table 3 and the target sequence of the PvB19 gene.
  • FIG. 7 is an explanatory diagram confirming the specificity between the probe candidates shown in Table 3 and the target sequence of the WNV gene.
  • FIG. 8 is an explanatory diagram of the arrangement of the probe set shown in Table 4 on the substrate of the microarray.
  • FIG. 9 is an explanatory view confirming the hybridization between the mixture of amplified products of each viral DNA amplified by PCR and the probe on the substrate of the microarray.
  • FIG. 10 is an explanatory view showing the detection sensitivity of the microarray according to the concentration of each viral DNA.
  • Viruses as detection targets of the present invention are five kinds of viruses of HIV, HCV, HBV, PvB19 and WNV, and specific detection targets are these viral genes.
  • the target sequences of these viral genes one or two of the HIV genes including the nucleotide sequence represented by SEQ ID NO: 11 (GenBank Accession No. NC_001802 from 4153th to 4464th) or a complementary nucleotide sequence thereof
  • SEQ ID NO: 11 GenBank Accession No. NC_001802 from 4153th to 4464th
  • oligonucleotides and HCV genes one or more oligonucleotides comprising the nucleotide sequence shown in SEQ ID NO: 12 (59th to 327th of GenBank Accession No.
  • HBV gene is one or two or more oligonucleotides comprising the nucleotide sequence represented by SEQ ID NO: 13 (450th to 715th GenBank Accession No. X — 70185) or its complementary nucleotide sequence, PvB1
  • the gene is a nucleotide sequence shown in SEQ ID NO: 14 (GenBank Accession No. NC — 000883, 2143 to 2278) or one or two or more oligonucleotides containing the complementary nucleotide sequence
  • the WNV gene is SEQ ID NO: 15 or 1 type or two or more types of oligonucleotides comprising the base sequence shown in 15 (GenBank Accession No.
  • NC — 009942, 3445th to 3599th or a complementary base sequence thereof. Even one type of these viruses or any combination of two or more types of viruses can be detected simultaneously. It is also possible to target a similar sequence of the target sequence, for example, a base sequence in which one or more base substitutions, deletions, insertions and additions or two or more types of mutations have occurred in the target sequence. Is possible.
  • the primer set for detecting the target sequence is composed of a primer consisting of the base sequence shown in SEQ ID NO: 1 and a primer consisting of the base sequence shown in SEQ ID NO: 2 for amplifying the HIV gene described in Table 1.
  • Primer set 1 for amplifying the HCV gene Primer set 2 consisting of a primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 4
  • Amplifying the HBV gene Primer set 3 composed of a primer consisting of the base sequence shown in SEQ ID NO: 5 and a primer consisting of the base sequence shown in SEQ ID NO: 6, and SEQ ID NO: 7 for amplifying the PvB19 gene
  • Primer comprising the base sequence and primer comprising the base sequence represented by SEQ ID NO: 8
  • primer set 5 composed of a primer consisting of the base sequence shown in SEQ ID NO: 9 and a primer consisting of the base sequence shown in SEQ ID NO: 10 for amplifying the W
  • Each nucleotide of the primer constituting the primer set can be chemically synthesized using, for example, a general-purpose DNA synthesizer. Oligonucleotides may be synthesized using any method known in the art. Oligonucleotides may be synthesized by commission.
  • the primer constituting the primer set of the present invention uses a fluorescently labeled primer so that the amplification region can be identified in order to detect a target sequence of a viral gene in a microarray described later.
  • the fluorescent labeling is preferably performed with Cy3 or Cy5, but the labeling method is not limited to this, and for example, a method using a labeled nucleotide as a substrate for PCR may be used.
  • a labeling method for example, when preparing a PCR amplification product, it is prepared by preparing ATCG4 types of deoxynucleotides (dNTPs) and labeled deoxynucleotides as substrates, and aligning the final concentration of each dNTP. A method of incorporating labeled deoxynucleotides into the amplified product is used.
  • PCR used in the present invention is performed according to a conventionally known technique.
  • the primer set in the present invention it is possible to amplify the target sequence of interest.
  • DNA is extracted from a biological sample for detection of target sequences of five types of viral genes in a biological sample, and this is used as a template DNA for PCR using the primer set of the present invention.
  • a biological sample for example, blood transfusion can be used.
  • a DNA extraction method a technique known in the art can be used. For example, extraction with phenol / chloroform or extraction using a commercially available DNA extraction reagent can be performed.
  • the primer set of the present invention is designed to amplify target sequences of five types of viral genes, it can be used to detect the expression of each viral gene in a biological sample.
  • RNA is extracted from a sample to prepare a template cDNA, and this is used as a template DNA to perform PCR using the primer set of the present invention and expressed in a biological sample. Amplify the target sequence of each gene.
  • RNA extraction method a technique known in the art can be used. For example, guanidine-isothiocyanate and phenol / chloroform extraction, or extraction using a commercially available RNA extraction reagent can be performed.
  • RNA as a template techniques known in the art can be used.
  • a commercially available random primer can be used as a primer, and cDNA can be prepared using reverse transcriptase in the presence of dNTPs.
  • the reverse transcriptase for example, SuperScript III (registered trademark) can be used.
  • PCR is performed using the primer set of the present invention using DNA or cDNA prepared as described above as template DNA.
  • a DNA polymerase for example, an amplification product can be obtained using GoTaq DNA Polymerase (registered trademark) or the like.
  • GoTaq DNA Polymerase registered trademark
  • the PCR method is well known in the art, and a primer that hybridizes to the sense strand (reverse primer) and a primer that hybridizes to the antisense strand (forward primer) are used.
  • reverse primer reverse primer
  • forward primer primer that hybridizes to the antisense strand
  • These primers, template DNA and DNA It can be carried out by repeating a cycle comprising annealing, extension and denaturation steps about 30 to 50 times in the presence of a polymerase.
  • PCR was performed at 95 ° C. for 2 minutes, then at 95 ° C. for 10 seconds, 55 ° C. for 10 seconds, 72 ° C. for 30 seconds, and finally at 72 ° C. for 7 minutes. did.
  • Probe set The probe for detecting the target sequence amplified by the primer set is described in Table 4, A probe consisting of the base sequence shown by SEQ ID NO: 16 (probe 1), a probe consisting of the base sequence shown by SEQ ID NO: 17 (probe 2), and the base sequence shown by SEQ ID NO: 18 for detecting the HIV gene Probe (probe 3), probe (probe 4) comprising the base sequence represented by SEQ ID NO: 19, probe comprising the base sequence represented by SEQ ID NO: 20 (probe 5), probe comprising the base sequence represented by SEQ ID NO: 21 (probe) Probe set 1 comprising a probe (probe 7) consisting of a base sequence represented by SEQ ID NO: 22; For detecting the HCV gene, a probe (probe 8) comprising the base sequence represented by SEQ ID NO: 23, a probe comprising the base sequence represented by SEQ ID NO: 24 (probe 9), and a base sequence represented by SEQ ID NO: 25 Probe (probe 10), probe (probe 11) comprising the
  • the target sequences of the five types of viral genes are simultaneously specified. Can be detected.
  • the probes constituting the probe set are designed to have a relatively short length of about 30 to 40 bp and have no sequence highly homologous to other viral DNAs.
  • the presence of each virus in the sample by hybridizing the PCR amplification product to a microarray in which probes specific for each virus DNA are immobilized on the substrate. Or the absence can be detected.
  • Each nucleotide of the primer constituting the probe set can be chemically synthesized using, for example, a general-purpose DNA synthesizer. Oligonucleotides may be synthesized using any method known in the art. Oligonucleotides may be synthesized by commission.
  • the probe set of the present invention is preferably used in the form of a microarray by being fixed on a substrate.
  • a method for producing the microarray a method in which an oligonucleotide prepared in advance is immobilized on the substrate surface is used.
  • the probe is covalently bonded to the surface-treated substrate via a spacer or a crosslinker.
  • the substrate include a nylon film, a nitrocellulose film, glass, and a silicon chip.
  • the probe can be fixed according to any method used in the art, but for example, the DNA may be fixed on the substrate using a spotter or the like.
  • Hybridization can be performed by dropping a fluorescently labeled DNA aqueous solution onto the microarray. Hybridization is preferably performed in the temperature range of room temperature to 70 ° C. for 0.5 to 20 hours. After completion of hybridization, washing is performed using a mixed solution of a surfactant and a buffer solution to remove unreacted labeled DNA. For detection of the fluorescent label, the presence of the amplified product can be detected by measuring the fluorescent signal intensity using, for example, a fluorescent scanner. In this embodiment, Gene Silicon (registered trademark) made of a 3 mm square silicon substrate is used as the microarray substrate, but the present invention is not limited to this.
  • the primer and the microarray could detect the target sequence of each virus gene prepared from the standard sample specifically and with high sensitivity.
  • the specificity of the primer was confirmed for each virus subtype gene.
  • one of the subtype genes of each virus was individually amplified by PCR, and then detection by microarray was confirmed using a mixture of each virus gene amplification product.
  • the target viral DNA needs to be highly specific in order to detect the target viral DNA from the presence of a large amount of viral and microbial DNA.
  • the primer set shown in Table 1 was selected, and primers were obtained by commissioned synthesis based on the sequence of the primer set shown in Table 1.
  • FIG. 1 is a flowchart for explaining the design of a target base sequence common degenerate primer targeting the target sequence of each viral gene.
  • the target base sequence common degenerate primer refers to a primer that can specifically amplify the target sequence of each virus subtype.
  • a sequence name is given to two types of two-base patterns (Gapped Tetra-Nucleotide Motif: GTNM) with a single base gap in between, and the sequence name and position are made into a database on the database.
  • GTNM Geled Tetra-Nucleotide Motif
  • the sequence as a common degenerate primer is determined (S5), the degenerate primer sequence and GTNM are linked, and depending on the binding temperature with the template DNA
  • the sequence length is adjusted for each primer as appropriate, and certified as a primary primer candidate (S6).
  • the three-dimensional structure of the base sequence recognized as the primary primer candidate is predicted, and the primer candidate of the base sequence forming the self-dimer and hairpin structure is excluded, and the rest is set as the secondary primer candidate (S7).
  • the base sequence forming the primer dimer is excluded from the secondary primer candidates (S8), and the sequence of the degenerate primer common to the target base sequence is obtained.
  • each lane of the agarose gel shows the genes of the subtypes of each of the viruses described above, and the order is from the left to the DNA molecular weight marker, and shows the genes of the subtypes in the above order. From this result, it was confirmed that the primer can specifically amplify the viral gene for each virus subtype gene.
  • Example preparation HIV-RNA ACCURUN 315 HIV-RNA positive Quality control (Subtype B, 2.1 ⁇ 10 5 Copies / ml, SeraCare) 47.62 ⁇ l was adjusted with defibrinated plasma (Basematrix 53, SeraCare), 1.0 ⁇ 10 4 cp / Reaction It was.
  • HCV-RNA ACCURUN 306 HCV-RNA positive Quality control (Subtype 1b, 9.1 ⁇ 10 5 IU / ml, SeraCare) 10.99 ⁇ l was adjusted with defibrinated plasma (Basematrix 53, SeraCare) and 1.0 ⁇ 10 4 IU / Reaction It was.
  • HBV-DNA ACCURUN 325 HBV-DNA positive Quality control (Subtype A, 8.1 ⁇ 10 6 Copies / ml, SeraCare) Adjust 12.35 ⁇ l with defibrinated plasma (Basematrix 53, SeraCare), 1.0 ⁇ 10 5 cp / Reaction It was.
  • PvB19-DNA Parvovirus B19 NATtrrol controls (Strain B19, 1.0 ⁇ 10 5 IU / ml, ZeptoMetrix) was adjusted with physiological saline (Otsuka Pharmaceutical) to obtain 2.0 ⁇ 10 4 IU / Reaction.
  • WNV-RNA West Nile virus NATtrrol controls (Strain NY 2001-6263, 5.0 ⁇ 10 4 Copies / ml, ZeptoMetrix) were adjusted with physiological saline (Otsuka Pharmaceutical) to obtain 1.0 ⁇ 10 4 cp / Reaction.
  • Each virus gene was recovered in a 50 ⁇ l Elution buffer from a standard sample (about 200 ⁇ l) of each virus gene using High Pure Viral Nucleic Acid Kit (for DNA extraction) or High Pure Viral RNA Kit (for RNA extraction).
  • the conditions for preparing cDNA are as follows.
  • PCR PCR reaction conditions performed using the template DNA of each virus prepared above were 95 ° C for 2 minutes, then 95 ° C for 10 seconds, 55 ° C for 10 seconds, and 72 ° C for 30 seconds for 50 cycles. Finally, PCR was performed at 72 ° C. for 7 minutes.
  • the probe set shown in Table 4 that is, a probe comprising the nucleotide sequence represented by SEQ ID NO: 16 (probe 1) for detecting the HIV gene, the nucleotide sequence represented by SEQ ID NO: 17
  • a probe comprising the nucleotide sequence represented by SEQ ID NO: 18 (probe 3), a probe comprising the nucleotide sequence represented by SEQ ID NO: 19 (probe 4), and a nucleotide sequence represented by SEQ ID NO: 20 that is, a probe comprising the nucleotide sequence represented by SEQ ID NO: 16 (probe 1) for detecting the HIV gene, the nucleotide sequence represented by SEQ ID NO: 17
  • a nucleotide sequence represented by SEQ ID NO: 20 that is, a probe comprising the nucleo
  • a probe set 1 comprising a probe (probe 5), a probe (probe 6) comprising a base sequence represented by SEQ ID NO: 21, and a probe (probe 7) comprising a base sequence represented by SEQ ID NO: 22,
  • a probe (probe 8) comprising the base sequence represented by SEQ ID NO: 23
  • a probe comprising the base sequence represented by SEQ ID NO: 24 (probe 9)
  • Probe (probe 10) comprising the base sequence represented by SEQ ID NO: 26
  • probe comprising the base sequence represented by SEQ ID NO: 27 (probe 12)
  • Probe set 2 comprising a probe (probe 14) consisting of a base sequence represented by SEQ ID NO: 29,
  • it consists of a probe consisting of the base sequence shown by SEQ ID NO: 30 (probe 15), a probe consisting of the base sequence shown by SEQ ID NO:
  • the probe set prepared above was arranged on a microarray as shown in FIG. 8A.
  • the probe was fixed to the silicon substrate using a spotter as shown in FIG. 8B to obtain a microarray.
  • the microarray substrate Gene Silicon (registered trademark) made of a 3 mm square silicon substrate was used.
  • Each virus DNA prepared from the standard sample was amplified using specific primers, mixed, dropped onto the microarray, baked at 80 ° C. for 1 hour, and then 2 ⁇ SSC / 0.2% SDS solution (95 For 5 minutes.
  • the probe was fixed on the microarray by rinsing with ultrapure water three times, removing water with a centrifuge and drying.
  • the fluorescence intensity (hybridization signal) of the spot was measured using a microarray scanner.
  • the measurement result of the obtained hybridization signal for each spot is shown in FIG. 9A.
  • FIG. 9A The measurement result of the obtained hybridization signal for each spot is shown in FIG. 9A.
  • FIGS. 9B to 9F show the results obtained when hybridization was carried out by dropping the mixture excluding each viral DNA onto the microarray when mixing the amplified products. This result also showed that the target sequence of each viral gene was detected specifically and with high sensitivity.
  • FIG. 10 shows the microarray detection sensitivity according to the concentration of each viral DNA. It was possible to detect HIV at 10 cp / Reaction, HCV at 5 IU / Reaction, HBV at 2 cp / Reaction, PvB19 at 2 IU / Reaction, and WNV at 5 cp / Reaction. From the above, it was shown that detection was possible with high sensitivity even at low concentrations.
  • the present invention is comprehensive with high specificity and sensitivity in a single detection operation for five types of viruses, such as HIV, HCV, HBV, PvB19 and WNV, having multiple genotypes in a biological sample, for example, blood transfusion.
  • viruses such as HIV, HCV, HBV, PvB19 and WNV
  • a biological sample for example, blood transfusion.

Landscapes

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

Abstract

L'invention concerne un procédé de détection complète de cinq types de virus VIH, VHC, VHB, PvB19 et WNV ayant chacun de multiples génotypes présentant une spécificité élevée et une sensibilité élevée, et des ensembles d'amorces, des puces à ADN et un nécessaire destinés à être utilisés dans la détection des virus. Chaque VIH, VHC, VHB, PvB19 et WNV ayant chacun de multiples génotypes peut être détecté par l'amplification d'un gène de chaque VIH, VHC, VHB, PvB19 et WNV par PCR en utilisant une amorce spécifique au génotype et par l'hybridation d'un produit d'amplification de PCR à l'aide d'une puce à ADN. À l'aide des ensembles d'amorces et de sondes pour détecter des virus, cinq types de virus VIH, VHC, VHB, PvB19 et WNV, possédant chacun de multiples génotypes, peuvent être détectés avec une spécificité élevée et une sensibilité élevée.
PCT/JP2010/006687 2009-12-14 2010-11-15 Procédé de détection complète de cinq types de virus vih, vhc, vhb, pvb19 et wnv ayant chacun de multiples génotypes, ensembles d'amorces, puces à adn et nécessaire de détection des virus WO2011074181A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009-283366 2009-12-14
JP2009283366A JP2011120556A (ja) 2009-12-14 2009-12-14 複数の遺伝子型を有するＨＩＶ、ＨＣＶ、ＨＢＶ、ＰｖＢ１９及びＷＮＶの５種類のウイルスの網羅的な検出方法、ウイルス検出用プライマーセット、マイクロアレイ及びウイルス検出用キット

Publications (1)

Publication Number Publication Date
WO2011074181A1 true WO2011074181A1 (fr) 2011-06-23

Family

ID=44166956

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/006687 WO2011074181A1 (fr) 2009-12-14 2010-11-15 Procédé de détection complète de cinq types de virus vih, vhc, vhb, pvb19 et wnv ayant chacun de multiples génotypes, ensembles d'amorces, puces à adn et nécessaire de détection des virus

Country Status (2)

Country Link
JP (1) JP2011120556A (fr)
WO (1) WO2011074181A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103421890A (zh) * 2013-03-14 2013-12-04 华中农业大学 牛源性肉制品分子鉴定试剂盒及应用
US20140275240A1 (en) * 2013-03-16 2014-09-18 Robert Benson Aylor Suppression and prevention of tumors and treatment of viruses
EP2809784A4 (fr) * 2012-01-31 2015-07-15 Advanced Liquid Logic Inc Amorces d'amplification et sondes pour détecter le vih-1

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2722397B1 (fr) * 2012-10-18 2017-12-13 F. Hoffmann-La Roche AG Analyse de sonde double pour la détection de populations d'amplicons hétérogènes
US11274998B2 (en) 2012-12-26 2022-03-15 Ventana Medical Systems, Inc. Specimen processing systems and methods for holding slides

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087361A1 (fr) * 2002-04-18 2003-10-23 Hokkaido Technology Licensing Office Co., Ltd. Procede de selection d'amorce pour synthese d'adn

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087361A1 (fr) * 2002-04-18 2003-10-23 Hokkaido Technology Licensing Office Co., Ltd. Procede de selection d'amorce pour synthese d'adn

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAIJI ENDO ET AL.: "Kyotsu Oligomer Bunseki ni Motozuku Tashu Virus Kyotsu Primer no Sekkei", JOURNAL OF VETERINARY MEDICINE, vol. 62, no. 19, 1 October 2009 (2009-10-01), pages 823 - 824 *
DAIJI ENDO: "Virus Kabu Kan Kyotsu Primer o Mochiita Shinraisei no Takai Virus Kenshutsu Hoho no Kento", MINISTRY OF HEALTH, LABOUR AND WELFARE KAGAKU KENKYUHI HOJOKIN, 24 April 2009 (2009-04-24), pages 21 - 71 *
KAZUSHIGE YAMAGUCHI ET AL.: "Kyotsu Motif Shugo primer Yosoku Algorithm (CoCoMo) o Mochiita HIV, HCV Oyobi HBV-yo degenerate primer no Sekkei", KOSEI RODO KAGAKU KENKYUHI HOJOKIN, 24 April 2009 (2009-04-24), pages 5 - 49 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2809784A4 (fr) * 2012-01-31 2015-07-15 Advanced Liquid Logic Inc Amorces d'amplification et sondes pour détecter le vih-1
CN103421890A (zh) * 2013-03-14 2013-12-04 华中农业大学 牛源性肉制品分子鉴定试剂盒及应用
US20140275240A1 (en) * 2013-03-16 2014-09-18 Robert Benson Aylor Suppression and prevention of tumors and treatment of viruses
US10292963B2 (en) * 2013-03-16 2019-05-21 Robert Benson Aylor Suppression and treatment of viruses

Also Published As

Publication number Publication date
JP2011120556A (ja) 2011-06-23

Similar Documents

Publication Publication Date Title
JPH07503143A (ja) Ｈｃｖ単離物のタイピング法
JP2000512160A (ja) Ｈｉｖ−１およびｈｉｖ−２を検出するための核酸プライマーおよびプローブ
JP2006502747A5 (fr)
JP7015288B2 (ja) シングルプレックス又はマルチプレックスアッセイにおいてヒトパルボウイルス核酸を検出及びａ型肝炎ウイルス核酸を検出するための組成物及び方法
WO2011074181A1 (fr) Procédé de détection complète de cinq types de virus vih, vhc, vhb, pvb19 et wnv ayant chacun de multiples génotypes, ensembles d'amorces, puces à adn et nécessaire de détection des virus
TW201221650A (en) Method for detection of enterovirus
JP2022546443A (ja) ｃｃｃＤＮＡから転写されたＨＢＶ ＲＮＡを含む、Ｂ型肝炎ウイルスＲＮＡの増幅および検出のための組成物および方法
KR20190002245A (ko) 지카바이러스 2중 진단키트 및 진단방법
ES2336807T3 (es) Deteccion del tipo y subtipo del vih.
CN101487064B (zh) 检测五种人兽共患病病毒的方法及专用试剂盒
JP2015231387A (ja) ヒトインターフェロンアルファサブタイプを検出するための組成物および使用方法
JP6592697B2 (ja) 血清型２のデングウイルス検出用キット、血清型３のデングウイルス検出用キット及びデングウイルス検出用キット
US11987851B2 (en) Reagents and methods for detecting HCV
JP2012143185A (ja) 口蹄疫ウィルスの包括的検出方法
WO2015150083A1 (fr) Procédé de détection spécifique du virus de la peste porcine classique
CN110914459A (zh) 用于检测乙型肝炎病毒(hbv)的测定
KR102332856B1 (ko) E형 간염 바이러스를 검출하기 위한 등온증폭 반응용 프라이머 세트 및 이의 용도
Chan et al. A comprehensive HBV array for the detection of HBV mutants and genotype
JP2018121607A (ja) ジカウイルス検出用プライマーセット、アッセイキットおよび増幅方法
CN106011309A (zh) 用于检测人丙型肝炎病毒的引物组和探针
CN107034312B (zh) 核苷酸组合物、试剂盒及其用途
JP2014140367A (ja) Ｂ型肝炎ウイルスｄｎａ増幅用プライマーおよびそれを用いたｂ型肝炎ウイルスｄｎａ検出法
WO2015001068A1 (fr) Amorces et procédés pour détecter des variants du virus de l'hépatite c (vhc) dans un échantillon isolé
JP6286904B2 (ja) ＳＮＰ（ｒｓ８１０３１４２）を検出するためのプローブ
AU2005201742B2 (en) Detection and identification of enteroviruses

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: 10837218

Country of ref document: EP

Kind code of ref document: A1

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

Ref document number: 10837218

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: 10837218

Country of ref document: EP

Kind code of ref document: A1