WO2013080307A1 - Jeu d'amorces pour amplifier les virus transmis par les moustiques, kit de dosage pour détecter les virus transmis par les moustiques, et procédé de détection faisant appel audit jeu d'amorces et audit kit de dosage - Google Patents

Jeu d'amorces pour amplifier les virus transmis par les moustiques, kit de dosage pour détecter les virus transmis par les moustiques, et procédé de détection faisant appel audit jeu d'amorces et audit kit de dosage Download PDF

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WO2013080307A1
WO2013080307A1 PCT/JP2011/077574 JP2011077574W WO2013080307A1 WO 2013080307 A1 WO2013080307 A1 WO 2013080307A1 JP 2011077574 W JP2011077574 W JP 2011077574W WO 2013080307 A1 WO2013080307 A1 WO 2013080307A1
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primer
virus
seq
nucleic acid
acid probe
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PCT/JP2011/077574
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English (en)
Japanese (ja)
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高橋　匡慶
源間　信弘
二階堂　勝
山本　直樹
美彩子 矢島
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株式会社 東芝
シンガポール国立大学
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Priority to PCT/JP2011/077574 priority Critical patent/WO2013080307A1/fr
Publication of WO2013080307A1 publication Critical patent/WO2013080307A1/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
    • C12Q1/70—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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a primer set for amplifying mosquito medium virus, an assay kit for detecting mosquito medium virus, and a detection method using them.
  • chikungunya virus dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus as the causative viruses of mosquito-borne infections. Since these viruses are also mosquito-borne viruses, it is desirable to detect them in a short time at a time.
  • a detection kit in order to comprehensively detect chikungunya virus, dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus, there are a plurality of different types of amplification devices for performing different amplification reactions. In addition, different detection reactions need to be performed.
  • the embodiment relates to a primer set for comprehensively amplifying mosquito-borne viruses.
  • the mosquito-borne viruses of interest are chikungunya virus, dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus.
  • the primer set includes a first primer group including SEQ ID NOS: 1 to 7, a second primer group including SEQ ID NOS: 8 to 12, a third primer group including SEQ ID NOS: 13 to 18, and SEQ ID NO: 19
  • a fourth primer group comprising -23, a fifth primer group comprising SEQ ID NOs: 24-29, a sixth primer group comprising SEQ ID NOs: 30-35, and a seventh primer comprising SEQ ID NOs: 36-41
  • a primer set for comprehensive amplification of chikungunya, dengue fever, West Nile fever, Japanese encephalitis and yellow fever is provided.
  • an assay kit capable of comprehensively detecting Chikungunya, Dengue fever, West Nile fever, Japanese encephalitis and yellow fever is provided.
  • a method is provided that can comprehensively detect Chikungunya, Dengue fever, West Nile fever, Japanese encephalitis and yellow fever.
  • Chikungunya virus, dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus amplification Chikungunya virus, Dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus are mosquito-borne viruses. In order to detect these viruses comprehensively, first, these viruses are comprehensively amplified.
  • the first primer group comprises a primer having SEQ ID NO: 1, a primer having SEQ ID NO: 2, a primer having SEQ ID NO: 3, a primer having SEQ ID NO: 4, a primer having SEQ ID NO: 5, a primer having SEQ ID NO: 6, a sequence A primer having the number 7 is included.
  • Sequence number 1 is CAGATGCGGTATGAGCCCTGTATGGAGAAGTCCGAATCATGC.
  • Sequence number 2 is CCGATGCGGTGTGGGCTCTGTATAGAGAAATCTGAATCTTGC.
  • SEQ ID NO: 1 and SEQ ID NO: 2 are FIP primers.
  • Sequence number 3 is TCCGCGTCCTTTACCAAGGAAATTTGGCGTCCTTAACTGTGAC and is a BIP primer.
  • Sequence number 4 is ACGCAATTGAGCGAAGCAC and is F3 primer.
  • Sequence number 5 is CTGAAGACATTGGCCCCAC and is a B3 primer.
  • SEQ ID NO: 6 is GCTGATGCAAATTCTGT, which is an LPF primer.
  • Sequence number 7 is CCTATGCAAACGGCGAC and is an LPB primer.
  • Dengue virus Dengue virus includes type 1, type 2, type 3 and type 4. In order to amplify the dengue virus exhaustively, each of these types uses an appropriate primer set.
  • Dengue virus type I (DENV-1) is comprehensively amplified by LAMP amplification using the second primer group.
  • the second primer group includes a primer having SEQ ID NO: 8, a primer having SEQ ID NO: 9, a primer having SEQ ID NO: 10, a primer having SEQ ID NO: 11, and a primer having SEQ ID NO: 12.
  • Sequence number 8 is GCTGCGTTGTGTCTTGGGAGGTTTTCTGTACGCATGGGGTAGC and is a FIP primer.
  • Sequence number 9 is CCCAACACCAGGGGAAGCTGTTTTTTTGTTGTTGTGCGGGGG and is a BIP primer.
  • Sequence number 10 is GAGGCTGCAAACCATGGAA and is F3 primer.
  • Sequence number 11 is CAGCAGGATCTCTGGTCTCT and is a B3 primer.
  • Sequence number 12 is GGTGGTAAGGACTAGAGG and is an LPB primer.
  • Dengue virus type II (DENV-2) is comprehensively amplified by LAMP amplification using the third primer group.
  • the third primer group includes a primer having SEQ ID NO: 13, a primer having SEQ ID NO: 14, a primer having SEQ ID NO: 15, a primer having SEQ ID NO: 16, a primer having SEQ ID NO: 17, and a primer having SEQ ID NO: 18.
  • Sequence number 13 is TTGGGCCCCCATTGTTGCTGTTTTAGTGGACTAGCGGTTAGAGG and is a FIP primer.
  • SEQ ID NO: 14 is GGTTAGAGGAGACCCCCCCAATTTTGGAGACAGCAGGATCTCTGG, which is a BIP primer.
  • SEQ ID NO: 15 is TGGAAGCTGTACGCATGG and is an F3 primer.
  • Sequence number 16 is GTGCCTGGAATGATGCTG and is a B3 primer.
  • SEQ ID NO: 17 is GATCTGTAAGGGAGGGG and is an LPF primer.
  • SEQ ID NO: 18 is GCATATTGACGCTGGGA, which is an LPB primer.
  • Dengue virus type III (DENV-3) is comprehensively amplified by LAMP amplification using the fourth primer group.
  • the fourth primer group includes a primer having SEQ ID NO: 19, a primer having SEQ ID NO: 20, a primer having SEQ ID NO: 21, a primer having SEQ ID NO: 22, and a primer having SEQ ID NO: 23.
  • SEQ ID NO: 19 is TGGCTTTTGGGCCTGACTTCTTTTTTGAAGAAGCTGTGCAGCCTG, which is a FIP primer.
  • Sequence number 20 is CTGTAGCTCCGTCGTGGGGATTTTCTAGTCTGCTACACCGTGC and is a BIP primer.
  • Sequence number 21 is GCCACCTTAAGCCACAGTA and is F3 primer.
  • Sequence number 22 is GTTGTGTCATGGGAGGG and is a B3 primer.
  • Sequence number 23 is GGAGGCTGCAAACCGTG and is an LPB primer.
  • Dengue virus type IV (DENV-4) is comprehensively amplified by LAMP amplification using the fifth primer group.
  • the fifth primer group includes a primer having SEQ ID NO: 24, a primer having SEQ ID NO: 25, a primer having SEQ ID NO: 26, a primer having SEQ ID NO: 27, a primer having SEQ ID NO: 28, and a primer having SEQ ID NO: 29 .
  • Sequence number 24 is TGGGAATTATAACGCCTCCCGTTTTTTCCACGGCTTGAGCAAACC and is a FIP primer.
  • Sequence number 25 is GGTTAGAGGAGACCCCTCCCTTTTAGCTTCCTCCTGGCTTCG and is a BIP primer.
  • SEQ ID NO: 26 is CTATTGAAGTCAGGCCAC and is an F3 primer.
  • Sequence number 27 is ACCTCTAGTCCTTCCACC and is a B3 primer.
  • Sequence number 28 is GGCGGAGCTACAGGCAG and is a LPF primer.
  • SEQ ID NO: 29 is TCACCAACAAAACGCAG and is an LPB primer.
  • the sixth primer group includes a primer having SEQ ID NO: 30, a primer having SEQ ID NO: 31, a primer having SEQ ID NO: 32, a primer having SEQ ID NO: 23, a primer having SEQ ID NO: 34, and a primer having SEQ ID NO: 35 .
  • Sequence number 30 is GCGGACGTCCAATGTTGGTTTGGCCACTTGGGTGGACTTG and is a FIP primer.
  • Sequence number 31 is AAGCTAGCCAACTTGCTGAGGTCGTCGAGATGTCAGTGACTG and is a BIP primer.
  • SEQ ID NO: 32 is GGAATGGGCAATCGTGACT and is an F3 primer.
  • Sequence number 33 is CGTTGTGAGCTTCTCCAGT and is a B3 primer.
  • SEQ ID NO: 34 is TCGTTTGCCATGATTGTC, which is an LPF primer.
  • Sequence number 35 is GAAGTTACTGCTATCATGCT and is an LPB primer.
  • the seventh primer group includes a primer having SEQ ID NO: 36, a primer having SEQ ID NO: 37, a primer having SEQ ID NO: 38, a primer having SEQ ID NO: 39, a primer having SEQ ID NO: 40, and a primer having SEQ ID NO: 41 .
  • SEQ ID NO: 36 is TTGGCCGCCTCCATATTCATCATTTTCAGCTGCGTGACTATCATGT, which is a FIP primer.
  • Sequence number 37 is TGCTATTTGGCTACCGTCAGCGTTTTTGAGCTTCTCCCATGGTCG and is a BIP primer.
  • SEQ ID NO: 38 is TGGATTTGGTTCTCGAAGG and is an F3 primer.
  • SEQ ID NO: 39 is GGTCAGCACGTTTGTCATT and is a B3 primer.
  • SEQ ID NO: 40 is CATCGATGGTAGGCTTGTC, which is an LPF primer.
  • SEQ ID NO: 41 is TTCCCACCAAAGCTGCGT, which is an LPB primer.
  • the eighth primer group includes a primer having SEQ ID NO: 42, a primer having SEQ ID NO: 43, a primer having SEQ ID NO: 44, a primer having SEQ ID NO: 45, a primer having SEQ ID NO: 46, and a primer having SEQ ID NO: 47 .
  • Sequence number 42 is CGGCTTCCCTTTGCTTTCCCAAAGGTGTCGGACTTGTGTGT and is a FIP primer.
  • Sequence number 43 is GGTATATGTGGCTGGGAGCGCCCCAATGGTCCTCATTCAGG and is a BIP primer.
  • SEQ ID NO: 44 is AAGGAAGCTGCACCAACAA and is an F3 primer.
  • SEQ ID NO: 45 is CTTCCACTCCTCCTCCTGA, which is a B3 primer.
  • SEQ ID NO: 46 is CTCTGACAGCTTCTTCTC, which is an LPF primer.
  • SEQ ID NO: 47 is GTATCTTGAGTTTGAGG and is an LPB primer.
  • Primer set for comprehensive amplification of mosquito-borne viruses consisting of chikungunya virus, dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus are as follows: A first primer set, a second primer set, a third primer set, a fourth primer set, a fifth primer set, a sixth primer set, a seventh primer set, and an eighth primer set are included.
  • Chikungunya virus, Dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus are comprehensively amplified.
  • Chikungunya virus, Dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus are amplified through the same amplification reaction using the same type of amplification device.
  • Chikungunya virus, dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus can be amplified easily and rapidly.
  • Chikungunya virus, dengue virus, Japanese encephalitis virus, West Nile virus and yellow fever virus can be simultaneously amplified, for example, in one reaction field.
  • Table 1 summarizes the primer sets for comprehensive amplification.
  • the comprehensive amplification primer set is a primer set for LAMP or RT-LAMP amplification.
  • the “LAMP method” and the “RT-LAMP method” are nucleic acid amplification methods. These are also referred to as isothermal gene amplification methods. Nucleic acids are amplified using DNA as a template by the LAMP method.
  • the RT-LAMP method amplifies a nucleic acid using RNA as a template by simultaneously performing a reverse transcription reaction and a LAMP reaction.
  • the LAMP method will be described, the RT-LAMP method can be performed in the same manner as the LAMP method except that a reverse transcription reaction is performed.
  • the LAMP method uses 2 or 4 region primers or 4 or 6 region primers.
  • the LAMP method is superior in amplification efficiency to the method using PCR and is not easily affected by impurities in the sample. Therefore, by using the primer according to this embodiment, it is possible to detect microorganisms classified as trace amounts of mosquito-borne viruses by simple sample pretreatment.
  • FIG. 1 shows double-stranded DNA to be detected in the center.
  • a total of four types of primer sequences that is, FIP primer, F3 primer, BIP primer, and B3 primer are set.
  • the FIP primer and the BIP primer each contain two regions (FIP is F1c and F2, and BIP is B2 and B1c).
  • the F3 primer contains the F3 region.
  • the B3 primer contains the B3 region.
  • the F3, F2, F1, B1c, B2c, and B3c regions are regions set in this order from the 5 ′ to 3 ′ direction of the single-stranded DNA of the double-stranded DNA.
  • the B3, B2, B1, F1c, F2c, and F3c regions are regions set in this order from the complementary strand to the 5 'to 3' direction of the single-stranded DNA.
  • B3 and B3c, B2 and B2c, B1 and B1c, F1c and F1, F2c and F2, and F3c and F3 are complementary strands.
  • a loop primer may be used in combination.
  • the loop primer is an LPF primer and / or an LPB primer.
  • amplification product having a dumbbell-shaped stem and loop structure as shown in FIG. 2 is obtained from each strand of the double-stranded DNA of FIG.
  • the structure shown in FIG. 2 is an example of the structure of some nucleic acids contained in the amplification product.
  • the description of the amplification mechanism is omitted, but if necessary, refer to, for example, Japanese Patent Laid-Open No. 2002-186871.
  • the RT-LAMP method is a method in which an enzyme having reverse transcription activity is added to the LAMP method reaction solution and the LAMP amplification method is performed while performing the reverse transcription reaction.
  • the same primer as the LAMP method can be used.
  • LAMP method when “LAMP method” is described, it may be understood that it is a reference to both the LAMP method and the RT-LAMP method.
  • the primer is designed so that the target sequence is located in the single-stranded loop part.
  • a “target sequence” is a sequence to be hybridized to a nucleic acid probe.
  • the target array (for example, any one of FPc, FP, BP, and BPc in FIG. 1) is between the regions F1 and F2 (this region may include the F2 region), and the region F2c Between F1c (this region may include F2c region), between regions B1 and B2 (this region may include B2 region), and / or between regions B2c and B1c (this region is B2c
  • Six primer regions are set so as to correspond to any of the regions (which may include a region).
  • the target sequence may be designed to be located at any part of the single-stranded loop portion formed between the respective regions.
  • the F2 region itself may be included in the loop portion between the primer regions F1 and F2.
  • Four types of primers are prepared based on the six primer regions thus set, and RT-LAMP amplification is performed using these primers. Thereby, a part of the LAMP amplification product as shown in FIG. 3 is obtained.
  • the target sequences FPc, FP, BP, and BPc are located in a single-stranded loop in the dumbbell structure of the amplification product.
  • the primer regions F1c and F1 and B1c and B1 originally have complementary sequences to each other, self-hybridization occurs with each other to form a double strand.
  • the target sequence contained in the amplification product exists in a single-stranded state as shown in FIG. Therefore, specific hybridization with a nucleic acid probe complementary to each target sequence (for example, a nucleic acid having a sequence of FP, FPc, BP, BPc) is possible without performing a denaturation operation, as shown in the figure. .
  • Detection of the amplification product can also be performed by using such a nucleic acid probe.
  • the amplification reaction may be performed using one primer set of the first to eighth primer sets per reaction field, for example, per tube, or the first reaction field (for example, one tube). It may be performed using at least two primer sets selected from the first to eighth primer sets, or may be performed using all of the first to eighth primer sets simultaneously.
  • the reaction field is where the reaction takes place.
  • the reaction field may be maintained inside a container in which the reaction can take place.
  • Such containers may be, for example, tubes, microtubes, beakers, multiwell plates, etc., but are not limited thereto.
  • the amplification reaction is not limited to this.
  • the amplification reaction is performed in a buffer having the following composition.
  • sample means body fluids, tissues, cells such as humans, mice, rats, guinea pigs, hamsters, ferrets, rhesus monkeys, rabbits, dogs, cats, cows, pigs, sheep, seals, and porpoises, and porpoises. It may be any substance present in the environment such as stool, insects such as mosquitoes, and water, lake water, river water, sea water and soil. Alternatively, the sample may be a substance suspected of containing a microorganism classified as a mosquito-borne virus or a gene thereof. For example, a culture medium may be used. Such a substance may be pretreated by means known per se into a state suitable for nucleic acid amplification, and any pretreatment may not be performed. Examples of such pretreatment include denaturation, filtration, nucleic acid extraction, impurity removal and the like.
  • Nucleic acid probe set The nucleic acid derived from mosquito-borne virus in the amplification product obtained by amplification as described above is specifically detected using the nucleic acid probe set.
  • the nucleic acid probe set has a nucleic acid probe having SEQ ID NO: 48, a nucleic acid probe having SEQ ID NO: 49, a nucleic acid probe having SEQ ID NO: 50, a nucleic acid probe having SEQ ID NO: 51, a nucleic acid probe having SEQ ID NO: 52, and a sequence number 53 A nucleic acid probe, a nucleic acid probe having SEQ ID NO: 54, a nucleic acid probe having SEQ ID NO: 55 and a nucleic acid probe having SEQ ID NO: 56.
  • SEQ ID NO: 48 is GCGACCATGCCGTCACAGTTAAGGA
  • SEQ ID NO: 49 is GCGATCATGCCGTCACAGTAAAGGA
  • SEQ ID NO: 50 is TAACCACTAGTCTGCTACCCCATGCGTACA
  • SEQ ID NO: 51 is ATTGACGCTGGGAAAGACCAGAGATCCTGC
  • SEQ ID NO: 52 is CCTTTGACGGGCATCGC 54 is CTATCTCCTTCTAGCACCAAGTCCACCCAA
  • SEQ ID NO: 55 is TAGGCTTGTCCTTAGACATGATAGTCACGC
  • SEQ ID NO: 56 is TCTTTTCCCCATCATGTTGTACACACAAGT.
  • the nucleic acid probes having SEQ ID NOs: 48 and 49 are nucleic acid probes for specifically detecting chikungunya virus.
  • the nucleic acid probe having SEQ ID NO: 50 is a nucleic acid probe for specifically detecting dengue virus type I.
  • the nucleic acid probe having SEQ ID NO: 51 is a nucleic acid probe for specifically detecting dengue virus type II.
  • the nucleic acid probe having SEQ ID NO: 52 is a nucleic acid probe for specifically detecting dengue virus type III.
  • the nucleic acid probe having SEQ ID NO: 53 is a nucleic acid probe for specifically detecting dengue virus type IV.
  • the nucleic acid probe having SEQ ID NO: 54 is a nucleic acid probe for specifically detecting Japanese encephalitis virus.
  • the nucleic acid probe having SEQ ID NO: 55 is a nucleic acid probe for specifically detecting West Nile virus.
  • the nucleic acid probe having SEQ ID NO: 56 is a nucleic acid probe for specifically detecting yellow fever virus.
  • a sample and first to eighth primer groups are added to a container for maintaining the reaction field (a). These are subjected to LAMP amplification under appropriate reaction conditions (b). Next, the obtained amplification product is added to a nucleic acid probe set containing nucleic acid probes having SEQ ID NOs: 45 to 56 (c), and a reaction is carried out (d).
  • FIG. 4 shows an example in which these nucleic acid probe sets are immobilized on a nucleic acid probe-immobilized substrate.
  • hybridization between the amplification product and each nucleic acid probe contained in the nucleic acid probe set is detected. For example, hybridization is detected by detecting a hybridization signal (e). Determine whether and / or how much mosquito-borne virus is present in the sample based on the presence or absence and / or intensity of the resulting hybridisation signal (F). By such a protocol, the method for detecting a mosquito-borne virus in a sample is implemented.
  • Means for detecting hybridization between the nucleic acid probe and the amplification product are not particularly limited to these, but turbidity, visible light, fluorescence, chemiluminescence, electrochemiluminescence, chemiluminescence, fluorescence energy transfer method
  • An optical method such as ESR, or electrical properties such as current, voltage, frequency, conductivity, and resistance may be used.
  • nucleic acid probe-immobilized chip is an apparatus in which the above-described nucleic acid probe is immobilized on a substrate that is a solid phase. This is generally called a microarray or a DNA chip. However, the nucleic acid probe is not immobilized on the chip and may be used in the liquid phase.
  • Nucleic acid probe immobilization chip can be used as a means for detecting hybridization between a nucleic acid probe and an amplification product.
  • the nucleic acid probe immobilization chip is an apparatus in which the above-described nucleic acid probe is immobilized on a substrate that is a solid phase. This is generally called a microarray or a DNA chip.
  • the nucleic acid probe immobilization chip 51 includes an immobilization region 53 on a base 52.
  • the nucleic acid probe 54 is immobilized on the immobilization region 53.
  • Such a nucleic acid probe immobilization chip 51 can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number of the fixing regions 53 arranged on the base 52 and the arrangement thereof as necessary.
  • Nucleic acid probes each having SEQ ID NO: 48 to SEQ ID NO: 56 as the nucleic acid probe 54 are immobilized in different immobilization regions 53 for each type.
  • the immobilization region 53 obtains a positive control region 55 for obtaining a positive control signal and / or a background signal in addition to the region for immobilizing the detection nucleic acid probe.
  • a background area 56 may be further provided.
  • the positive control region 55 and the background region 56 may be configured to obtain a positive signal and a background signal by using any technique known per se.
  • Such a nucleic acid probe-immobilized chip may be suitably used for a detection method using fluorescence.
  • one type or one or more types of nucleic acid probes or nucleic acid probe sets necessary for obtaining information that needs to be obtained as one independent signal are immobilized.
  • the nucleic acid probe immobilization chip 61 in FIG. 6 includes an electrode 63 as an immobilization region on a base 62.
  • the nucleic acid probe 64 is immobilized on the electrode 63.
  • the electrode 63 is connected to the pad 67. Electrical information from the electrode 63 is acquired via the pad 67.
  • Such a nucleic acid probe immobilization chip 61 can be manufactured by a method well known in the art.
  • the number of electrodes 63 arranged on the base 62 and the arrangement thereof can be appropriately changed by those skilled in the art as needed.
  • the nucleic acid probe immobilization chip 61 of this example may include a reference electrode and a counter electrode as necessary. In one immobilization region, one type or one or more types of nucleic acid probes or nucleic acid probe sets necessary for obtaining information that needs to be obtained as one independent signal are immobilized.
  • the nucleic acid probes each having SEQ ID NO: 48 to SEQ ID NO: 56 as the nucleic acid probe 64 are immobilized on different electrodes 63 for each type.
  • the nucleic acid probe immobilization chip 61 has an electrode 63 for obtaining a positive control electrode 65 for obtaining a positive control signal and / or a background signal in addition to the region for immobilizing the detection nucleic acid probe.
  • a background electrode 66 may be further provided.
  • the positive control region 55 and the background region 56 may be configured to obtain a positive signal and a background signal by using any technique known per se.
  • a functional group such as an amino group, a thiol group, or biotin may be introduced at the end of the nucleic acid probe described above.
  • a spacer may be introduced between the functional group and the nucleotide.
  • the substrate for immobilizing the nucleic acid probe used in this embodiment is not particularly limited, but includes resin beads, magnetic beads, metal fine particles, microtiter plates, glass substrates, silicon substrates, resin substrates, electrode substrates, and the like. May be used.
  • the substrate material used in this embodiment is not particularly limited.
  • inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
  • the electrode is not particularly limited, but, for example, simple metals such as gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium and tungsten, and alloys thereof, or graphite and glassy It may be carbon such as carbon, or an oxide or compound thereof. Further, a semiconductor compound such as silicon oxide, or various semiconductor devices such as CCD, FET, and CMOS may be used.
  • the extraction method is not particularly limited, and a liquid-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can be used. Further, a commercially available nucleic acid extraction method QIAamp (manufactured by QIAGEN), (manufactured by Co., Ltd.) or the like can also be used. Next, RT-LAMP amplification is performed using the extracted nucleic acid component as a template, and then a hybridization reaction is performed with the gene detection electrode. The reaction solution is carried out in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10.
  • dextran sulfate, salmon sperm DNA, bovine thymus DNA, EDTA and / or a surfactant, which are hybridization accelerators, can be added.
  • the extracted and amplified nucleic acid component is added to this and then donated to the hybridization reaction with the nucleic acid probe.
  • the reaction rate can be increased by an operation such as stirring or shaking.
  • the reaction temperature is in the range of 10 ° C to 90 ° C, and the reaction time is about 1 minute to overnight.
  • a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used for washing after the hybridization reaction.
  • the detection may be performed by using a second nucleic acid probe labeled with the aforementioned substance.
  • the detection is performed according to the following procedure. After the substrate is washed, a nucleic acid binding substance that selectively binds to the double-stranded portion formed on the electrode surface may be allowed to act to perform electrochemical measurement.
  • the nucleic acid binding substance used here is not particularly limited, and examples thereof include Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalators and bisintercalators such as bisacridine, trisintercalators and polyintercalators. May be used.
  • these intercalators can be modified with an electrochemically active metal complex such as ferrocene and viologen.
  • the concentration of the nucleic acid binding substance varies depending on the type, but is generally used in the range of 1 ng / ml to 1 mg / ml. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used. After the electrode is reacted with the nucleic acid binding substance, electrochemical measurement is performed. The electrochemical measurement is performed with a three-electrode type, that is, a reference electrode, a counter electrode, and a working electrode, or a two-electrode type, that is, a counter electrode and a working electrode.
  • a gene detection apparatus using a gene detection electrode includes a gene extraction unit, a gene reaction unit, a nucleic acid binding substance reaction unit, an electrochemical measurement unit, a washing unit, and the like.
  • An assay kit for comprehensively detecting mosquito-borne viruses includes the primer set and the nucleic acid probe set.
  • the assay kit may contain a buffer necessary for LAMP amplification and / or RT-LAMP amplification in addition to the primer set and nucleic acid probe set described above, and a buffer necessary for hybridization, and the amplification and / or high concentration therein.
  • a container for performing hybridization may be provided.
  • the nucleic acid probe contained in the assay kit may be provided immobilized on a substrate as described above, or the assay kit may be provided including a substrate for immobilizing a nucleic acid probe.
  • One or more primers contained in the first to eighth primer groups contained in the primer set contain mutations to the extent that the nucleic acid derived from the corresponding mosquito-borne virus can be specifically amplified. It may be an array.
  • the mutation is caused by substitution, deletion and / or substitution of 1 to 5, preferably 1 to several nucleotides at any position in the polynucleotide represented by the sequence described in any of the above SEQ ID NOs or a complementary sequence thereof. It may be an insertion.
  • the specifically amplifiable range may be a range that does not affect the identification when a mosquito-borne virus to be amplified by a primer is taxonomically identified.
  • a plurality of sequences containing such mutations may be simultaneously present in one amplification reaction field.
  • a complementary sequence thereof may be prepared as a mixed nucleotide.
  • 1 to 5, preferably 1 to several nucleotides at any position may be universal nucleotides.
  • universal nucleotides include, but are not limited to, deoxyinosine (dI), Gren Research 3-nitropyrrole, 5-nitroindole, deoxyribofuranosyl DP) and deoxy-5′-dimethoxytrityl-D-ribofuranosyl; dK) and the like.
  • a sequence of about 1 to 100 nucleotides, preferably about 2 to 30 nucleotides (for example, a sequence used as a spacer) may be included between each sequence of the primer or on the terminal side thereof.
  • the length of the primer may be about 30 to 200 nucleotides, preferably 35 to 100 nucleotides, more preferably 43 to 60 nucleotides.
  • each of the FIP primer, the BIP primer, the F3 plumer, and the B3 primer may be of one type, or each may contain a plurality of types of sequences.
  • the LPF primer and the LPB primer may each consist of one kind of sequence, and each may contain a plurality of kinds of sequences.
  • amplification by a LAMP method or RT-LAMP method is performed on a sample containing a nucleic acid derived from a mosquito-borne virus using a primer using the region as described above, as described in FIG.
  • a loop structure is formed, resulting in a target sequence derived from a mosquito-borne virus contained within a single-stranded loop structure.
  • This target sequence is a sequence for hybridizing the nucleic acid probe. Therefore, when at least one primer included in the primer set includes a mutation, the corresponding at least one nucleic acid probe included in the nucleic acid probe set may include a mutation accordingly.
  • primers In order to amplify mosquito-borne viruses more universally, a plurality of types of FIP primers, BIP primers, F3 plumer, B3 primer, LPF primer and LPB primer may be used. In order to amplify the virus more reliably and comprehensively, primers may be designed and used so that they are universal nucleotides or mixed bases at least with respect to mutation sites specific to species.
  • a plurality of sequences with different base types may be represented by a single sequence for the mutation part.
  • the base of the mutation part in question is described by including a single base symbol indicating a plurality of bases.
  • the meanings of the bases indicated by these symbols are those commonly used in the art.
  • r is guanine or adenine
  • y is thymine or cytosine
  • m is adenine or cytosine
  • k is guanine or thymine
  • s is guanine or cytosine
  • w is adenine or thymine
  • B is guanine or cytosine or thymine
  • d is adenine or guanine or thymine
  • h is adenine or cytosine or thymine
  • v is adenine or guanine or cytosine
  • n is adenine or guanine or cytosine or It is thymine.
  • each primer can be designed as follows.
  • the sequence of the region in each virus is downloaded from a known database such as the NCBI nucleotide database, and an alignment is created to clarify the mutation.
  • the primer corresponding to the mutation position is included in the primer as a mix base.
  • the polyprotein region of yellow fever virus can be downloaded from the NCBI database and an alignment can be created to obtain the conserved region.
  • the primer is then designed using, for example, the software “Primer Explorer V4” (see “Primer Explorer V4”, “http://primerexplorer.jp/”).
  • the designed primer may be synthesized by a method known per se.
  • accession numbers for each mosquito media virus sequence that can be used to design each primer are listed below. Primers containing mutations and mixed bases may be made by making alignments for these sequences.
  • nucleic acid probe set described above may include mutation in at least one nucleic acid probe contained therein.
  • the mutation contained in the nucleic acid probe may include a mutation within a range where the nucleic acid derived from the mosquito-borne virus can be specifically detected.
  • the specifically detectable range may be a range that does not affect the identification when the target mosquito-borne virus is taxonomically identified using a nucleic acid probe containing a mutation.
  • each of the above-described nucleic acid probes may be composed of a sequence in which one or several bases at any position in the sequence represented by the above SEQ ID No. are substituted, deleted, or inserted.
  • the nucleic acid probe is changed according to the genetic polymorphism or mutation held by each strain of mosquito-borne virus to be detected, and one or several bases at any position in the sequence indicated by the above SEQ ID No. It may consist of a sequence with substitutions, deletions or bases inserted. A plurality of sequences containing such mutations may be simultaneously present in one reaction field. In that case, 1 to 5, preferably 1 to several nucleotides at any position of the polynucleotide represented by the sequence described in any of the above SEQ ID Nos. Or a complementary sequence thereof may be prepared as a mixed nucleotide. In general, 1 to 5, preferably 1 to several nucleotides at any position may be universal nucleotides.
  • universal nucleotides include, but are not limited to, deoxyinosine (dI), Gren Research 3-nitropyrrole, 5-nitroindole, deoxyribofuranosyl DP) and deoxy-5′-dimethoxytrityl-D-ribofuranosyl; dK) and the like.
  • the nucleic acid probe may further contain a sequence of about 1 to 100 nucleotides, preferably about 2 to 30 nucleotides (for example, a sequence used as a spacer) on the terminal side.
  • the length of the nucleic acid probe may be about 30 to 200 nucleotides, preferably 35 to 100 nucleotides, and more preferably 43 to 60 nucleotides.
  • nucleic acid probes described above may be used alone or in multiple types in one reaction field. Moreover, when using it fixed to a base
  • the “mixed base” is a mixture of two or more nucleic acid probes designed as “adenine”, “thymine”, “cytosine”, and “guanine” as bases at desired positions to be mixed bases.
  • a nucleic acid probe set Moreover, you may design so that it may substitute with the modified base which can be paired with respect to multiple types of bases.
  • the mixed base may be used as a mixed base for a base at one position in one reaction field, or may be used as a mixed base corresponding to all of the bases at a plurality of positions.
  • the structure of the nucleic acid probe is not particularly limited, and DNA, RNA, PNA, LNA, methylphosphonate skeleton nucleic acid and / or other artificial nucleic acid chains can be used.
  • a chimeric nucleic acid of any of these nucleic acids may be used.
  • primer set Design of primer set for yellow fever virus Specifically, the polyprotein region of yellow fever virus was downloaded from the NCBI nucleotide database. An alignment was created to obtain the saved area. The primers were then designed using, for example, the software “Primer Explorer V4” (see “Primer Explorer V4”, “http://primerexplorer.jp/”). The designed primers are from Life Technologies Inc. , And requested the synthesis as a cartridge grade. These gave a primer group for yellow fever virus.
  • the prepared primer group includes a primer having SEQ ID NO: 42, a primer having SEQ ID NO: 43, a primer having SEQ ID NO: 44, a primer having SEQ ID NO: 45, a primer having SEQ ID NO: 46, and SEQ ID NO: 47. It was a primer.
  • Sequence number 42 is CGGCTTCCCTTTGCTTTCCCAAAGGTGTCGGACTTGTGTGT and is a FIP primer.
  • Sequence number 43 is GGTATATGTGGCTGGGAGCGCCCCAATGGTCCTCATTCAGG and is a BIP primer.
  • SEQ ID NO: 44 is AAGGAAGCTGCACCAACAA and is an F3 primer.
  • SEQ ID NO: 45 is CTTCCACTCCTCCTCCTGA, which is a B3 primer.
  • SEQ ID NO: 46 is CTCTGACAGCTTCTTCTC, which is an LPF primer.
  • SEQ ID NO: 47 is GTATCTTGAGTTTGAGG and is an LPB primer.
  • primer groups for chikungunya virus, dengue fever virus, Japanese encephalitis virus and west nile virus (1) chikungunya virus
  • a primer group for chikungunya virus was prepared.
  • an LPB primer having CCTATGCAAACGGCGAC of SEQ ID NO: 7 were prepared.
  • Dengue virus (a) Dengue virus type I Primers for dengue virus type I were prepared. FIP primer with GCTGCGTTGTGTCTTGGGAGGTTTTCTGTACGCATGGGGTAGC being SEQ ID NO: 8, BIP primer having CCCAACACCAGGGGAAGCTGTTTTTTTGTTGTTGTGCGGGGG being SEQ ID NO: 9, F3 primer having GAGGCTGCAAACCATGGAA being SEQ ID NO. An LPB primer having was prepared.
  • (B) Dengue virus type II Primers for dengue virus type II were prepared.
  • FIP primer having SEQ ID NO: 13 TTGGGCCCCCATTGTTGCTGTTTTAGTGGACTAGCGGTTAGAGG
  • BIP primer having GGTTAGAGGAGACCCCCCCAATTTTGGAGACAGCAGGATCTCTGG SEQ ID NO: 14
  • F3 primer having TGGAAGCTGTACGCATGG SEQ ID NO: 16
  • TGGCCTGAB TGGCCTGAB
  • an LPB primer having GCATATTGACGCTGGGA which is SEQ ID NO: 18.
  • (C) Dengue virus type III Primers for dengue virus type III were prepared.
  • FIP primer having TGGCTTTTGGGCCTGACTTCTTTTTTGAAGAAGCTGTGCAGCCTG which is SEQ ID NO: 19
  • BIP primer having CTGTAGCTCCGTCGTGGGGATTTTCTAGTCTGCTACACCGTGC having SEQ ID NO: 21
  • F3 primer having GCCACCTTAAGCCACAGTA having SEQ ID NO: 21
  • GTGGGTG having GTGGGTGCAT having GTG
  • (D) Dengue virus type IV Primers for dengue virus type IV were prepared.
  • FIP primer with SEQ ID NO: 24 TGGGAATTATAACGCCTCCCGTTTTTTCCACGGCTTGAGCAAACC
  • an LPB primer having TCACCAACAAAACGCAG which is SEQ ID NO: 29 were prepared.
  • Japanese encephalitis virus A primer group for Japanese encephalitis virus was prepared.
  • FIP primer having GCGGACGTCCAATGTTGGTTTGGCCACTTGGGTGGACTTG which is SEQ ID NO: 30
  • BIP primer having AAGGCTAGCCAACTTGCTGAGGTCGTCGAGATGTCAGTGACTG being SEQ ID NO31
  • F3 primer having GTATGGGTCTG3 which is GGTTTGTTGC having GGTTTG
  • an LPB primer having GAAGTTACTGCTATCATGCT which is SEQ ID NO: 35 were prepared.
  • plasmid DNA into which a nucleotide sequence region necessary for LAMP amplification reaction of each virus and a T7 promoter were introduced was synthesized.
  • the base sequence of the introduction part is as follows.
  • Dengue type III standard synthetic template DNA Standard synthetic template DNA for Dengue fever type III is shown in Table 4-4.
  • Dengue type IV standard synthetic template DNA Standard synthetic template DNA for dengue type IV is shown in Table 4-5.
  • Japanese encephalitis virus standard synthetic template DNA Standard synthetic template DNAs for Japanese encephalitis virus are shown in Table 4-6.
  • Yellow fever virus standard synthetic template DNA Standard synthetic template DNAs for yellow fever virus are shown in Table 4-8.
  • RNA was prepared from standard synthetic template DNA for each of the viruses described above. The synthesis of RNA from the standard synthetic template DNA was performed using T7 RiboMAX Express Large Scale RNA Production system (Promega). Transcribed RNA was purified by RNA easy (QIAGEN Inc.), and RNA concentration was measured using Qubit (Life Technologies Inc.). A standard synthetic template RNA was prepared by adjusting the RNA concentration to 1E + 04 copies / ⁇ L TE.
  • Reverse transcription-LAMP reaction Table 1 shows the sequences of primer sets used in the reverse transcription-LAMP reaction (RT-LAMP). These are primers synthesized in the preparation of the above primer set.
  • Table 2 shows the composition of reagents necessary for the reverse transcription-LAMP reaction.
  • a primer set not including LPF primer and LPB primer
  • sterilized distilled water (DW) was added correspondingly. This solution was reacted at 63 C ° for 50 minutes to carry out reverse transcription-LAMP reaction.
  • nucleic acid probe-immobilized chip A DNA chip was prepared as a nucleic acid probe-immobilized chip.
  • Table 3 shows the nucleotide sequence of the nucleic acid probe DNA immobilized on the DNA chip. Nucleic acid probes having the base sequences described in Table 3 were artificially synthesized.
  • the mosquito-borne virus detection kit of the present application can simultaneously detect five viruses and four serotypes of dengue fever.

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Abstract

Le présent mode de réalisation selon l'invention concerne un jeu d'amorces pour amplifier complètement un virus transmis par les moustiques. Le virus transmis par les moustiques à amplifier comprend le virus chikungunya, le virus de la dengue, le virus de l'encéphalite japonaise, le virus du Nil occidental et le virus de la fièvre jaune. Le jeu d'amorces comprend un premier groupe d'amorces comprenant les SEQ ID No : 1 à 7, un deuxième groupe d'amorces comprenant les SEQ ID No : 8 à 12, un troisième groupe d'amorces comprenant les SEQ ID No : 13 à 18, un quatrième groupe d'amorces comprenant les SEQ ID No : 19 à 23, un cinquième groupe d'amorces comprenant les SEQ ID No : 24 à 29, un sixième groupe d'amorces comprenant les SEQ ID No : 30 à 35, un septième groupe d'amorces comprenant les SEQ ID No : 36 à 41 et un huitième groupe d'amorces comprenant les SEQ ID No : 42 à 47.
PCT/JP2011/077574 2011-11-29 2011-11-29 Jeu d'amorces pour amplifier les virus transmis par les moustiques, kit de dosage pour détecter les virus transmis par les moustiques, et procédé de détection faisant appel audit jeu d'amorces et audit kit de dosage WO2013080307A1 (fr)

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WO2014039010A1 (fr) * 2012-09-04 2014-03-13 Republic Polytechnic Oligonucléotides isolés, procédés et trousses pour la détection, l'identification et/ou la quantitation des virus de chikungunya et de la dengue
WO2015072847A1 (fr) * 2013-11-14 2015-05-21 Universiti Malaya Procédé de détection et d'identification du sérotype de virus de la dengue
CN108707693A (zh) * 2018-02-13 2018-10-26 连云港出入境检验检疫局检验检疫综合技术中心（江苏国际旅行卫生保健中心连云港分中心、连云港出入境检验检疫局口岸门诊部） 一种黄热病毒、基孔肯雅病毒快速荧光pcr检测试剂盒
TWI723675B (zh) * 2019-12-12 2021-04-01 國立成功大學 用於檢測登革熱病毒的方法以及套組

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014039010A1 (fr) * 2012-09-04 2014-03-13 Republic Polytechnic Oligonucléotides isolés, procédés et trousses pour la détection, l'identification et/ou la quantitation des virus de chikungunya et de la dengue
WO2015072847A1 (fr) * 2013-11-14 2015-05-21 Universiti Malaya Procédé de détection et d'identification du sérotype de virus de la dengue
CN108707693A (zh) * 2018-02-13 2018-10-26 连云港出入境检验检疫局检验检疫综合技术中心（江苏国际旅行卫生保健中心连云港分中心、连云港出入境检验检疫局口岸门诊部） 一种黄热病毒、基孔肯雅病毒快速荧光pcr检测试剂盒
TWI723675B (zh) * 2019-12-12 2021-04-01 國立成功大學 用於檢測登革熱病毒的方法以及套組

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