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WO2018181703A1 - Procédé de détection multiplex pour 5 types de parasites paludéens - Google Patents

Procédé de détection multiplex pour 5 types de parasites paludéens Download PDF

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WO2018181703A1
WO2018181703A1 PCT/JP2018/013219 JP2018013219W WO2018181703A1 WO 2018181703 A1 WO2018181703 A1 WO 2018181703A1 JP 2018013219 W JP2018013219 W JP 2018013219W WO 2018181703 A1 WO2018181703 A1 WO 2018181703A1
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seq
nucleotide sequence
oligonucleotide
sequence
oligonucleotide represented
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真弘 平塚
雄大 齋藤
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国立大学法人東北大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for detecting five kinds of malaria parasites, a primer set for detecting malaria parasites, and a kit for detecting malaria parasites.
  • malaria parasites that infect humans include Plasmodium falciparum, Plasmodium vivax, Oval malaria (Plasmodium ovale), Plasmodium malariae and Salmalaria (Plasmodium). Five types of knowlesi) are known. Delays in the diagnosis of malaria infections lead to delays in treatment, and there are many cases of death after becoming severe. Therefore, the development of a simple, rapid, highly sensitive and species-specific malaria diagnostic method that can be carried out in malaria endemic areas has become an urgent issue internationally.
  • the test for malaria parasites is the gold standard, in which a blood smear is stained with Giemsa and observed with an optical microscope. This microscopic method can be confirmed or denied by a skilled person. However, if the density of protozoa is low, a long examination time and advanced techniques are required, and as a result, treatment is likely to be delayed. For this reason, a rapid diagnosis method for malaria (Rapid Diagnostic Test; RDT) using immunochromatography has been developed. However, the specificity of distinguishing malaria species is low, and false positives after infection recovery are a problem.
  • PCR is more sensitive than microscopy and RDT, and can be diagnosed with high specificity against mixed infections.
  • fluorescence detector in order to detect the final DNA amplification product, it is necessary to use an expensive fluorescence detector and perform complicated gel electrophoresis, which is useful for routine diagnosis in general hospitals and on-site clinics in malaria-endemic areas. Is not suitable.
  • Non-Patent Document 1 describes four types of malaria parasites of P. falciparum, T. falciparum malaria, oval malaria, and K. falciparum malaria by PCR targeting malaria parasite mitochondrial cytochrome c oxidase III (cox3). A detection method by PCR is described. However, this document does not include detection of salmalaria.
  • the object of the present invention is to provide five kinds of malaria (Plasmodium (falciparum), Plasmodium vivax, Oval malaria (Plasmodium ovale), Plasmodium malariae and Salmalaria (Plasmodium knowlesi).
  • the object is to provide a simple and rapid method for detecting malaria parasites.
  • the present inventors have determined that, based on the base sequence of mitochondrial cytochrome c oxidase III (cox3) of Plasmodium falciparum, P. falciparum malaria, S. falciparum malaria, oval malaria, A. malaria and monkeys.
  • Two or more types of oligonucleotide primers that can specifically identify each of the five malaria parasites of malaria are prepared, and DNA sequences characteristic of the five types of malaria parasites are PCR amplified using a primer set comprising such primers.
  • the presence or absence of malaria infection and the protozoan species of the infected malaria are visually detected and identified from the position of the detected band. Or found that it can be diagnosed.
  • Item 1 One or more detection methods selected from the group consisting of Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasite, and Salmonaria parasites present in a sample, (1) a step of amplifying a nucleic acid using the following five types of primer sets (a) to (e); and (2) a Plasmodium falciparum based on the nucleic acid amplified in step (1), A method comprising the step of detecting one or more selected from the group consisting of Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasite, and Salmonaria parasite.
  • A an oligonucleotide represented by the base sequence of SEQ ID NO: 1 or an oligonucleotide represented by a base sequence in which one or two bases are deleted, inserted or substituted in the base sequence of SEQ ID NO: 1, and tropical An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum, and one or two bases deleted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2
  • a primer set consisting of an oligonucleotide represented by an inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum
  • B an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or an oligonucleot
  • a primer set comprising an oligonucleotide represented by a deleted, inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax (C) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 5; An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P.
  • C an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 5;
  • a primer set comprising an oligonucleotide represented by a deleted, inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum (D) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 7, and an egg Oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium parasite and one or two bases deleted in the oligonucleotide represented by the nucleot
  • step (1) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 11 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 11, and plasmodium Oligonucleotide for obtaining an amplification product of a specific region of the genus cox3 gene sequence, and one or two bases deleted or inserted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence of SEQ ID NO: 12
  • a nucleic acid belonging to the genus Plasmodium is genus-specific using a primer set consisting of an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the Plasmodium cox3 gene sequence Am
  • Step (1) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 13 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 13, and plasmodium Oligonucleotide for obtaining an amplification product of a specific region of the genus cox3 gene sequence, and one or two bases deleted or inserted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 14 or the nucleotide sequence of SEQ ID NO: 14 Alternatively, using a primer set consisting of an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the Plasmodium cox3 gene sequence, the DNA obtained from the specimen is used as a template.
  • Item 4. The method according to Item 3, wherein the step of obtaining the primary amplification product and the step of obtaining the secondary amplification product are performed in a single reaction.
  • step (1) when the primer set (a) to (e) is present, (f) is present, the primer set (a) to (f) is present, or (g) is present Any one of Items 1-4, comprising amplifying the primer set of (a) to (e) and (g) or the primer set of (a) to (g) in a mixed state in one reaction solution The method according to claim 1.
  • each of the primer sets (a) to (e), or (f) when present, each of the primer sets (a) to (f) has a tag sequence attached to one oligonucleotide, Item 6.
  • Item 7 A method for detecting Plasmodium falciparum using a primer set comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2.
  • Item 8 An oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3, and C.
  • oligonucleotide for obtaining a specific region amplification product of protozoan cox3 gene sequence oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or nucleotide sequence of SEQ ID NO: 4 with one or two bases deleted or inserted
  • the protozoa of Plasmodium vivax using a primer set comprising an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax Detection method.
  • Item 9 A method for detecting Plasmodium falciparum using a primer set comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6.
  • Item 10 A method for detecting an oval malaria parasite using a primer set consisting of an oligonucleotide represented by the base sequence of SEQ ID NO: 7 and an oligonucleotide represented by the base sequence of SEQ ID NO: 8.
  • An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence and one or two bases deleted, inserted or substituted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 10 or the nucleotide sequence of SEQ ID NO: 10 A method for detecting a protozoan parasite using a primer set comprising an oligonucleotide represented by the determined nucleotide sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of the protozoan parasite.
  • a primer set for detecting Plasmodium falciparum detection comprising an oligonucleotide represented by the base sequence of SEQ ID NO: 1 and an oligonucleotide represented by the base sequence of SEQ ID NO: 2.
  • oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3, and C.
  • oligonucleotide for obtaining a specific region amplification product of protozoan cox3 gene sequence oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or nucleotide sequence of SEQ ID NO: 4 with one or two bases deleted or inserted
  • a primer set for detecting Plasmodium falciparum comprising an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum.
  • a primer set for detecting Plasmodium falciparum detection comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6.
  • a primer set for detecting an oval malaria parasite comprising an oligonucleotide represented by the base sequence of SEQ ID NO: 7 and an oligonucleotide represented by the base sequence of SEQ ID NO: 8.
  • a primer set for detection of protozoan malaria parasites which is an oligonucleotide represented by the determined base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of the protozoan parasite.
  • Item 17. The primer set according to any one of Items 12 to 16, wherein a tag sequence is attached to one oligonucleotide and a labeling substance or a labeling substance introduction material is attached to the other oligonucleotide.
  • Item 18 From the following 5 types of primer sets (a) to (e), DNA polymerase, and dNTP, from Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum and Plasmodium falciparum One or more detection kits selected from the group consisting of:
  • A an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or an oligonucleotide represented by a nucleotide sequence in which one base is deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 1, and Plasmodium falciparum
  • An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2 with one or two bases deleted, inserted or
  • a primer set comprising an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum
  • B an oligonucleotide represented by the nucleotide sequence of SEQ ID NO:
  • a primer set comprising an oligonucleotide represented by a deleted, inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax (C) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 5; An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P.
  • C an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 5;
  • a primer set comprising an oligonucleotide represented by a deleted, inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum (D) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 7, and an egg Oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium parasite and one or two bases deleted in the oligonucleotide represented by the nucleot
  • Item 21 When the primer set (a) to (e) is present, (f) is present (a) to (f), or (g) is present (a) to (e) ) And (g) or the primer sets (a) to (g) mixed in one reaction solution, the primer sets (a) to (e), Item 21.
  • each of the primer sets (a) to (e), or (f) when present, each of the primer sets (a) to (f) has a tag sequence attached to one oligonucleotide, Item 22.
  • a primer set comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NOs: 1 to 10 or an oligonucleotide represented by a nucleotide sequence in which one or two bases thereof are modified is used. Presence and type of five types of human malaria parasites, malaria parasites, Plasmodium falciparum, Plasmodium malaria parasites, egg-shaped malaria parasites, and simian malaria parasites, can be detected easily and rapidly.
  • FIG. 1 The top view of one Embodiment of the device for nucleic acid detection
  • FIG. 2 Side view. 2 is a schematic diagram showing a state in which the nucleic acid detection device of FIG. 1 is immersed in a test solution. Schematic which shows the state which covered the device for nucleic acid detection with the transparent exterior body. Schematic of malaria species specific PCR using primer set. Schematic for demonstrating malaria detection by nucleic acid chromatography. The photograph which shows the detection of the band in the strip 10 minutes after expansion
  • the detection method of the present invention comprises at least one selected from the group consisting of Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasites, and Salmonaria parasites present in a sample.
  • a detection method comprising: (1) a step of amplifying a nucleic acid using the following five types of primer sets (a) to (e); and (2) based on the nucleic acid amplified in step (1). And detecting one or more selected from the group consisting of Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasite, and Salmonaria parasite.
  • A an oligonucleotide represented by the base sequence of SEQ ID NO: 1 or an oligonucleotide represented by a base sequence in which one or two bases are deleted, inserted or substituted in the base sequence of SEQ ID NO: 1, and tropical An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum, and one or two bases deleted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2
  • a primer set comprising an oligonucleotide represented by an inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum
  • b a base of SEQ ID NO: 3
  • SEQ ID NO: 6 an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence of SEQ ID NO: 6 lacking one or two bases
  • SEQ ID NO: 7 comprising an oligonucleotide represented by a deleted, inserted or substituted nucleotide sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum Or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 7, and the cox3 gene of an oval malaria parasite An oligonucleotide for obtaining an amplification product of a specific region of the sequence; An oligonucleotide represented by the nucleotide sequence of No.
  • Each oligonucleotide in the primer sets (a) to (e) used in the detection method of the present invention targets the cytochrome c oxidase 3 gene (cox3) of mitochondrial DNA of plasmodium parasite.
  • Each oligonucleotide in one of the pair of primer sets (a) to (e) above has a sequence complementary to the base sequence of the cox3 gene of each malaria parasite, and each primer set corresponds to the corresponding malaria parasite. It is prepared to amplify a specific region of the cox3 gene. Therefore, five types of malaria parasites can be detected with higher sensitivity and higher efficiency than PCR based on the 18rDNA sequence, which has been considered a standard for PCR detection of malaria parasite DNA for a long time.
  • oligonucleotides (a) to (e) are designed so that one of the five types of malaria parasites can be specifically detected and no cross-reaction occurs. All primer sets can be mixed in a single reaction solution and amplified and detected in multiplex. Within 1.5 hours, preferably within 1 hour, the presence or absence of malaria infection and the identification or diagnosis of infected species can be performed quickly. It can be carried out.
  • PCR is performed by mixing all the primer sets used for PCR in one reaction solution, and the amplification product is spread on the strip, so that the presence or absence of infection with five types of malaria parasites is visually identified on one strip. can do.
  • the method using a nucleic acid chromatography strip can be performed at room temperature, it is not necessary to prepare an agarose gel at the time of use or to store it at a low temperature unlike the conventional method.
  • an expensive thermal cycler with a fluorescence detector is not required, five types of malaria parasites can be detected at a simple and low cost.
  • each oligonucleotide in the primer sets (a) to (e) used in the detection method of the present invention described above includes SEQ ID NO: 1
  • An oligonucleotide represented by a base sequence in which one or two bases are deleted, inserted or substituted in each base sequence of ⁇ 10, and an amplification product of a specific region of the cox3 gene sequence of five malaria parasites Also included are oligonucleotides to obtain. As long as each base sequence of SEQ ID NOs: 1 to 10 functions as a primer for obtaining an amplification product, one or two bases may be deleted, inserted or substituted.
  • SEQ ID NO: 12 For example, even if the first base from the 5 ′ end of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12 is deleted, Although the sensitivity is inferior to that in the case of no deletion, it has been found that genus-specific detection of each malaria parasite is possible. It is also well known in the art that substitution of one base usually has no effect on the pairing between a base sequence and its complementary sequence.
  • a nucleic acid belonging to the genus Plasmodium is genus-specific using a primer set consisting of an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the Plasmodium cox3 gene
  • a tag sequence may be attached to the end with or without a spacer.
  • an oligonucleotide represented by the base sequence of SEQ ID NO: 1 or a modified sequence thereof (b) an oligonucleotide represented by the base sequence of SEQ ID NO: 3 or a modified sequence thereof, and (c) a base of SEQ ID NO: 5
  • a tag sequence may be attached to the 5 ′ end of each of the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 11 in (f) or a modified sequence thereof.
  • the tag sequence is a sequence designed to eliminate cross reaction between tags regardless of the sequence of the target nucleic acid of 5 kinds of malaria adults, preferably 15 bases to 50 bases, more preferably 20 bases or more and 25 bases or less.
  • spacer and tag sequences are well known in the field of nucleic acid detection.
  • the spacer is a gene non-expression part consisting of a polymerase reaction inhibition region inserted between the primer and the tag sequence.
  • the spacer can include a nucleic acid derivative or a non-nucleic acid derivative.
  • Nucleic acid derivatives include L-type nucleic acids, 3-deoxy-2-hydroxy-dN, modified base nucleic acids, damaged base nucleic acids, phosphate binding site modified nucleic acids, RNA, 2′-OMe-N, and derivatives thereof. There may be mentioned at least one selected from the group.
  • the non-nucleic acid derivative is at least one selected from the group consisting of a carbon chain (C n ), a PEG chain ((CH 2 CH 2 O) n ), a disulfide-containing chain (C n SSC n ), and a dithiol phosphoramidite. One of them.
  • the above (a) to (e) and (f) above in order to facilitate detection after amplification of the target nucleic acid by PCR, the above (a) to (The other of the two oligonucleotides to be paired in each primer set of f) or both of the two oligonucleotides may be labeled with a labeling substance that can be observed with the naked eye, or a labeling substance-introducing material.
  • oligonucleotide represented by the base sequence of SEQ ID NO: 2 or a modified sequence thereof (b) the oligonucleotide represented by the base sequence of SEQ ID NO: 4 or a modified sequence thereof, and (c) the base of SEQ ID NO: 6
  • a labeling substance or a labeling substance-introducing material may be attached to the 5 ′ end of each of the oligonucleotide represented by the base sequence of SEQ ID NO: 12 in (f) or its modified sequence.
  • a tag sequence is attached to the 5 ′ end of the oligonucleotides represented by the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or their respective modified sequences.
  • the oligonucleotides represented by the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or their respective modified sequences are labeled at the 5 ′ end.
  • a substance or a labeling substance introducing material may be attached.
  • the labeling substance introduction material is a material for binding to the labeling substance, and includes, for example, biotin for binding to the labeling substance coated with avidin, and enables the labeling substance to be introduced into the target nucleic acid.
  • the labeling substance is preferably a luminescent substance or a coloring substance that presents luminescence or coloring that can be detected visually (with the naked eye).
  • labeling substances include chemiluminescent substances including various dyes, various pigments, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, aryl imidazoles, lucigenin, luciferin and eclion. It is done.
  • colloids or sols including gold colloids or sols or silver colloids or sols, metal particles, inorganic particles, and the like can be given.
  • the labeling substance may have particles such as latex particles in part.
  • the average particle diameter of particles such as latex particles constituting a part of the labeling substance is not particularly limited, but for example, 20 nm or more and 20 ⁇ m or less, preferably 0.1 ⁇ m or more and 10 ⁇ m or less, particularly preferably 0.1 ⁇ m or more and 5 ⁇ m or less, More preferably, they are 0.15 micrometer or more and 2 micrometers or less. Moreover, it adjusts suitably according to the hole diameter of a solid-phase carrier.
  • the particles are particles that can be suspended in an aqueous solution and are made of a water-insoluble polymeric material.
  • a water-insoluble polymeric material for example, polyethylene, polystyrene, styrene-styrene sulfonate copolymer, acrylic acid polymer, methacrylic acid polymer, acrylonitrile polymer, acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate, polyvinylpyrrolidone, or vinyl chloride-acrylate may be mentioned. Mention may also be made of latex particles having active groups on their surface, for example carboxyl, amino or aldehyde groups.
  • labeling substance introduction material examples include antibodies in antigen-antibody reaction, biotin in avidin (streptavidin) -biotin system, digoxigenin in anti-digoxigenin (DIG) -digoxigenin (DIG) system, or FITC in anti-FITC-FITC system Haptens and the like.
  • the labeling substance used for detection is the other molecule or substance that interacts with the labeling substance-introducing material (for example, antigen, ie, streptavidin, anti-FITC, etc.), and in the hybridization step with the oligonucleotide probe, or Prior to or after this step, the labeling substance introduction material attached to the amplification product and the labeling substance having a site that binds to the labeling substance introduction material are bound to enable detection of the amplification product.
  • the labeling substance-introducing material for example, antigen, ie, streptavidin, anti-FITC, etc.
  • the step (1) comprises (g) the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 13 or the nucleotide sequence of SEQ ID NO: 13 lacking one or two bases.
  • An oligonucleotide represented by a deleted, inserted or substituted nucleotide sequence, and an oligonucleotide for obtaining an amplification product of a specific region of the Plasmodium cox3 gene sequence and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 14
  • a primer set consisting of Using NA as a template to obtain a primary amplification product, and using the primer sets (a) to (e) above, or (f) the primer set (a) to (f), And a step of obtaining a secondary amplification product using the primary amplification product as a template.
  • Nested PCR detection sensitivity can be improved and noise during amplification can be avoided, so that five types of malaria parasites can be detected with higher sensitivity and specificity.
  • Nested PCR after performing a PCR reaction using the first primer set (primary amplification), the inner region of the amplified gene (amplified product) is replaced with the second primer set. In order to perform amplification (secondary amplification), it is necessary to perform PCR reaction twice.
  • a primer set for primary amplification (primer set of (g)), a primer set common to Plasmodium (primer set of (f)), and a species-specific primer set ((a) to ( Since the annealing temperature of the primer set (e) is different from each other and the annealing temperature of the species-specific primer set (primer set (a) to (e)) is the same,
  • the reaction conditions it is possible to achieve Nested PCR in one PCR reaction and to distinguish and detect five types of malaria parasites.
  • the PCR conditions in the present invention are not particularly limited as long as the target DNA fragment can be amplified to a detectable level.
  • the amount of double-stranded DNA as a template used in the PCR reaction is 0 ⁇ m. 1 ng or more is preferable, and 1 to 50 ng is more preferable.
  • a heat denaturation reaction for converting a double-stranded DNA into a single strand is performed at 92 to 98 ° C. for 10 to 60 seconds, and an annealing reaction for hybridizing the primer pair to the single-stranded DNA at 55 to 70 ° C. for 10 to 60 seconds.
  • An extension reaction for allowing DNA polymerase to act is performed at 71 to 75 ° C.
  • the denaturation temperature is preferably 92 to 98 ° C.
  • the denaturation time is preferably 30 seconds to 10 minutes.
  • the primary amplification is performed by heat denaturation reaction at 92 to 98 ° C. for 10 to 60 seconds, and annealing reaction at 65 to 70 ° C. for 10 to 60 seconds.
  • the extension reaction is carried out at 71 to 75 ° C. for 10 to 60 seconds, and these cycles are preferably carried out for 10 to 30 cycles.
  • the heat denaturation reaction is carried out at 92 to 98 ° C. for 10 to 60 cycles.
  • the annealing reaction is performed at a temperature lower than the annealing temperature of the primary reaction, for example, 55 to 65 ° C. for 10 to 60 seconds, and the extension reaction is performed at 71 to 75 ° C. for 10 to 60 seconds. Is preferably carried out for 20 to 40 cycles.
  • the present invention includes a method for detecting Plasmodium falciparum using a primer set consisting of an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2.
  • the step of amplifying the nucleic acid in the sample and the step of detecting P. falciparum based on the amplified nucleic acid are as described in the above step (1) and step (2). According to this method, P. falciparum can be detected species-specifically.
  • the present invention relates to an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3, and An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax, an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or one or two bases in the nucleotide sequence of SEQ ID NO: 4 Three days using a primer set comprising an oligonucleotide represented by a deleted, inserted or substituted nucleotide sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum A method for detecting a malaria parasite is included. The step
  • Plasmodium falciparum can be detected species-specifically.
  • the present invention includes a method for detecting Plasmodium falciparum using a primer set consisting of an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6.
  • the step of amplifying the nucleic acid in the sample and the step of detecting Plasmodium falciparum based on the amplified nucleic acid are as described in the above step (1) and step (2).
  • Plasmodium falciparum can be detected species-specifically.
  • the present invention includes a method for detecting an oval malaria parasite using a primer set consisting of an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 8.
  • the step of amplifying the nucleic acid in the sample and the step of detecting the oval malaria parasite based on the amplified nucleic acid are as described in the above step (1) and step (2).
  • the oval malaria parasite can be detected in a species-specific manner.
  • the present invention relates to an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 9, Oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax and one or two bases deleted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 10 or the nucleotide sequence of SEQ ID NO: 10
  • plasmodium parasite can be detected in a species-specific manner.
  • the present invention includes a primer set for detecting P. falciparum comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2.
  • Such a primer set can be used as a specific primer set for detecting Plasmodium falciparum.
  • the present invention includes a primer set for detecting P. falciparum comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2.
  • the present invention relates to an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3, and
  • An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or one or two bases in the nucleotide sequence of SEQ ID NO: 4
  • For detection of Plasmodium falciparum comprising an oligonucleotide represented by a deleted, inserted or substituted nucleotide sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum Includes primer set.
  • Such a primer set can be used as a specific primer set for detecting Plasmodium falciparum.
  • the present invention encompasses a primer set for detecting Plasmodium falciparum detection comprising an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6.
  • Such a primer set can be used as a specific primer set for detecting Plasmodium falciparum.
  • the present invention includes a primer set for detecting an oval malaria parasite comprising an oligonucleotide represented by the base sequence of SEQ ID NO: 7 and an oligonucleotide represented by the base sequence of SEQ ID NO: 8.
  • Such a primer set can be used as a specific primer set for detecting oval malaria parasites.
  • the present invention relates to an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 9, Oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax and one or two bases deleted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 10 or the nucleotide sequence of SEQ ID NO: 10 And a primer set for detection of Plasmodium vivax, which is an oligonucleotide represented by an inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium vivax.
  • Such a primer set can be used as a specific primer set for detecting Plasmodium vivax.
  • an oligonucleotide represented by the base sequence of SEQ ID NO: 3 or an oligonucleotide represented by a base sequence in which one or two bases are deleted, inserted or substituted in the base sequence of SEQ ID NO: 3, and for 3 days An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum, and one or two bases deleted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 4
  • the above primer set may be used in a PCR method, or may be used in a LAMP method, which is a gene amplification method for amplifying a nucleic acid at a constant temperature, an SDA method, an ICAN method, or an RCA method.
  • the present invention further includes the following five types of primer sets (a) to (e), a DNA polymerase, dNTP, and a reaction buffer solution: Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Egg It includes one or more detection kits selected from the group consisting of protozoan malaria parasites and simian malaria parasites.
  • A an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or an oligonucleotide represented by a nucleotide sequence in which one base is deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 1, and Plasmodium falciparum
  • An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2 with one or two bases deleted, inserted or
  • a primer set comprising an oligonucleotide represented by a substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum (b) represented by the base sequence of SEQ ID NO: 3 1 or 2 in the oligonucleot
  • the DNA polymerase may be any template-dependent nucleic acid synthase having strand displacement activity.
  • the polymerases include Bst DNA polymerase (large fragment), Bca (exo-) DN A polymerase, Klenow fragment of E. coli DNA polymerase I, Vent (Exo-) DNA polymerase (excluding exonuclease activity from Vent DNA polymerase) ), DeepVent (Exo-) DNA polymerase (DeepVent DNA polymerase excluding exonuclease activity), KOD DNA polymerase, and the like.
  • DNTP includes dATP, dTTP, dGTP, and dCTP.
  • reaction buffer examples include Tris buffer, EDTA buffer, and Tris-EDTA buffer.
  • the detection kit comprises (f) an oligonucleotide represented by the base sequence of SEQ ID NO: 11 or a base in which one or two bases are deleted, inserted or substituted in the base sequence of SEQ ID NO: 11 And an oligonucleotide for obtaining an amplification product of a specific region of the Plasmodium cox3 gene sequence, and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence of SEQ ID NO: 12 A primer set consisting of an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted, and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium In addition.
  • the genus-specific detection of the genus Plasmodium can be performed simultaneously with the species-specific detection of the five types of Plasmodium with one kit.
  • the detection kit can perform amplification and detection in a state where the five types of primer sets (a) to (e) and optionally the primer set (f) are mixed in one reaction solution.
  • the presence or absence of malaria infection and the identification or diagnosis of the infected species can be performed quickly.
  • the detection kit includes (g) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence of SEQ ID NO: 13, wherein one or two bases are deleted, inserted or substituted
  • An oligonucleotide represented by the base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium and the oligonucleotide represented by the base sequence of SEQ ID NO: 14 or the base sequence of SEQ ID NO: 14
  • a primer set comprising an oligonucleotide represented by a nucleotide sequence in which one or two bases are deleted, inserted or substituted, and an oligonucleotide for obtaining an amplification product of a specific region of a Plasmodium cox3 gene sequence Further included.
  • the primer set for primary amplification (primer set of (g)), the primer set common to Plasmodium (primer set of (f)), and the species-specific primer set ( Designed so that the annealing temperatures of the primer sets (a) to (e) are different and the annealing temperatures of the species-specific primer sets (primers of (a) to (e)) are the same. Therefore, by devising the reaction conditions, Nested PCR can be achieved in one PCR reaction, and five types of malaria parasites can be distinguished and detected.
  • the target nucleic acid sequences of the five types of Plasmodium are oligos having a base sequence that can hybridize with a tag sequence attached to one of two pairs of oligonucleotides in each of the primer sets (a) to (f) above. Detection can be performed by a nucleic acid detection device provided with a solid phase carrier holding a nucleotide probe.
  • FIG. 1 (A) is a plan view of an embodiment of a nucleic acid detection device
  • FIG. 1 (B) is a side view of the nucleic acid detection device.
  • the nucleic acid detection device 1 of one embodiment includes a solid phase carrier 10.
  • the solid phase carrier 10 has a porous material that holds an oligonucleotide probe for detecting a target nucleic acid of a malaria parasite to be detected.
  • the “oligonucleotide probe” is an oligonucleotide having a sequence complementary to the sequence of the DNA tag, which can hybridize to the DNA tag attached to the oligonucleotide primer for amplification of the target nucleic acid of the malaria parasite to be detected. It can also be a nucleotide, or it can be an oligonucleotide designed to have a sequence that can specifically hybridize with a malaria parasite target nucleic acid.
  • the length of the oligonucleotide sequence of the oligonucleotide probe is not particularly limited, but is preferably 15 bases or more and 50 bases or less in order to ensure specificity to the hybridizing nucleic acid and hybridization efficiency.
  • Porous materials are well known in the art, and examples include cellulose, nitrocellulose, and nylon.
  • the solid support 10 is usually formed in a strip shape or a sheet shape.
  • the length of the oligonucleotide probe sequence is not particularly limited, but is preferably 15 bases or more and 50 bases or less in order to ensure specificity and hybridization efficiency for each target nucleic acid.
  • the nucleic acid detection device 1 may include a backing member 12 that supports the solid phase carrier 10. Furthermore, optionally, the nucleic acid detection device 1 prevents the gas from flowing between the solid phase carrier 10 and the external environment, and maintains the airtightness of the hybridization reaction environment between the oligonucleotide probe and the target nucleic acid. May be provided.
  • the covering member 14 is a transparent film that covers the surface of the solid phase carrier, and the material constituting the transparent film is not particularly limited as long as it maintains the airtightness of the surface of the solid phase carrier and prevents evaporation of the developing solution. Examples thereof include polyethylene terephthalate. By providing the covering member 14, it is possible to easily suppress the generation of non-specific signals (non-specific hybridization) in nucleic acid chromatography and to detect the target nucleic acid with higher sensitivity.
  • oligonucleotide fixing regions 16a, 16b, 16c, 16d, 16e, and 16f are positions where oligonucleotide probes are fixed on the solid phase carrier 10, and from the front end of the solid phase carrier 10.
  • the target nucleic acids corresponding to the target nucleic acids are fixed in parallel to each other at a certain distance.
  • the method for immobilizing the oligonucleotide probe to the solid phase carrier 10 is not particularly limited, and may be bound to the solid phase carrier 10 at the 3 'end or may be bound to the 5' end.
  • the fixing property is enhanced by UV irradiation or the like.
  • the probe is immobilized only on the surface of the solid-phase carrier 10 where UV reaches.
  • one or a plurality of position markers 18a, 18b, 18c may be arranged so that a probe region corresponding to the target nucleic acid can be easily specified. Due to the presence of the position markers 18a, 18b, and 18c, the presence or absence of the target nucleic acid can be easily detected even in the case of visual detection.
  • nucleic acid detection using the nucleic acid detection device 1 using a sample amplified by PCR will be described as an example.
  • a specific region of the cox3 gene sequence of the malaria parasite is amplified from the DNA in the sample, and the sample containing the target nucleic acid is amplified by the first primer and the second primer. Amplify using the primer set.
  • the first primer has a first identification sequence and a tag sequence that are complementary to the target nucleic acid and identify the first base sequence in the target nucleic acid, and the tag sequence and the first identification sequence are directly linked. It is preferable to have a linking site capable of suppressing or stopping the DNA polymerase reaction between them.
  • This linking site does not contain a natural base or a derivative of a natural base (such as a natural base) that pairs with a natural base, and is composed of, for example, an artificial oligonucleotide, and thus suppresses or stops the DNA polymerase reaction.
  • a nucleic acid having a single-stranded region is amplified, and hybridization with an oligonucleotide probe becomes possible without thermal denaturation.
  • the second primer paired with the first primer in PCR is complementary to the target nucleic acid, in addition to the second identification sequence for identifying the second base sequence in the target nucleic acid, and the amplified target nucleic acid is an oligonucleotide. It includes a labeling substance that can be visually confirmed when hybridized with the probe, or a labeling substance-introducing material.
  • the labeling substance and the labeling substance introduction material are as described above.
  • test solution containing the amplification product after amplifying a specific region of the cox3 gene sequence of Plasmodium from the DNA in the sample is applied to the tip of the solid phase carrier 10.
  • the applied test solution is developed in the porous material constituting the solid phase carrier 10 by a capillary phenomenon.
  • the tip of the solid phase carrier 10 can be developed on the solid phase carrier 10 by immersing the tip of the solid phase carrier 10 in a test solution 22 containing the amplified target nucleic acid placed in a container 20 such as an Eppendorf tube. .
  • the solid phase carrier 10 is entirely covered, and the tip of the solid phase carrier 10 is disposed near the tip 32 of the outer packaging member 30, at least a part of which is transparent.
  • the test solution 22 in the container 20 can be put into the exterior member 30 via the sample port 34 provided at the tip of the part 32 and the tip of the solid carrier 10 can be immersed in the solid carrier 10.
  • the exterior member 30 must be at least partially transparent so that the target nucleic acid can be visually detected.
  • the test solution preferably contains a “developing medium” so as to facilitate the development of the target nucleic acid in the solid phase carrier 10.
  • the development medium is not particularly limited, and examples thereof include water, an organic solvent compatible with water, or a mixture of water and one or more organic solvents.
  • the organic solvent compatible with water include lower alcohols having about 1 to 4 carbon atoms, esters such as DMSO, DMF, methyl acetate, and ethyl acetate, acetone, and the like.
  • the development medium is preferably mainly water.
  • the development medium can contain a buffer component for adjusting the pH.
  • the buffer component is usually in the range of 6.0 to 8.0, depending on the intended pH. More preferably, it is 7.0 or more and 8.0 or less.
  • Components for obtaining such pH are, for example, acetic acid and sodium acetate (acetic acid buffer), citric acid and sodium citrate (citrate buffer), phosphoric acid and sodium phosphate (phosphate buffer), and the like.
  • a phosphate buffered saline (PBS) etc. are mentioned.
  • composition and concentration may be adjusted by adding an additional component such as a surfactant or an appropriate salt or a solvent to the amplification reaction solution, and the resultant may be used as a developing medium.
  • the development time is not particularly limited, and is appropriately set according to the form and shape of the solid phase carrier 10 and the properties of the development medium.
  • the oligonucleotide probe held on the solid phase carrier 10 has a sequence complementary to the sequence of the DNA tag capable of hybridizing to the DNA tag attached to the first primer, it hybridizes with the first primer, Since one primer hybridizes with a second primer containing a labeling substance or a labeling substance-introducing material, a plurality of target nucleic acids can be visually observed in a species-specific and / genus-specific manner by color development of a line on which an oligonucleotide probe is immobilized. It can be identified and detected.
  • Multiplex PCR Table 1 shows the nucleotide sequences of the oligonucleotide primers used in the multiplex PCR for detecting the five species of Plasmodium plasmodium.
  • MtPuni_F SEQ ID NO: 11
  • MtPuni_R SEQ ID NO: 12
  • 5 species-specific primers of P. falciparum, S. falciparum malaria, egg-shaped malaria, A. vivax malaria, and sal malaria were designed based on the cox3 gene sequence of each malaria parasite.
  • MtPf_F SEQ ID NO: 1
  • MtPf_R SEQ ID NO: 2
  • MtPv_F (SEQ ID NO: 3) and MtPv_ R (SEQ ID NO: 4) are three days respectively.
  • a forward primer and a reverse primer specific for heat malaria and MtPm_F (SEQ ID NO: 5) and MtPm_ ⁇ R (SEQ ID NO: 6) are respectively a forward primer and a reverse primer specific for P. falciparum malaria
  • MtPk_F (SEQ ID NO: 9) and MtPk_ R (SEQ ID NO: 10) are monkeys, respectively.
  • Forward and reverse primers specific for malaria are monkeys, respectively.
  • Each PCR primer is linked to a cox3 gene sequence characteristic of each malaria species, followed by a spacer (three carbon linkages) and a DNA tag sequence.
  • DNA tags 1, 2, 4, 3, 7, and 8 are attached to the 5 ′ ends of the forward primers consisting of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, respectively.
  • Biotin was added to the 5 ′ end of the reverse primer consisting of the base sequences of SEQ ID NOs: 2, 4, 6, 8, 10, and 12 paired with each of them.
  • a forward primer MtU_F (SEQ ID NO: 1) and a reverse primer MtU_R (SEQ ID NO: 2) for primary amplification were also designed.
  • Table 2 shows the amounts of reagents and primers used for PCR. Reagents and all primers were mixed in one reaction solution, placed in PCR 0.2 ml tube band separate 8 series natural (Greiner Bio-One) and subjected to PCR.
  • Table 3 shows the PCR conditions.
  • a T100 thermal cycler Bio-Rad
  • Nested PCR can be performed in one reaction in one tube by setting the reaction temperature to three stages.
  • denaturation was performed once at 95 ° C. for 3 minutes (initial denaturation reaction).
  • primary amplification with primary amplification primers MtU_F and MtU_R was performed. Specifically, 10 cycles of denaturation 95 ° C., 15 seconds, annealing 70 ° C., 30 seconds, elongation 72 ° C., 30 seconds were performed. This amplification amplifies the common sequence of the five malaria species.
  • genus-specific primers MtPuni_F and MtPuni_R and 5 types of malaria parasite-specific primers MtPf_F, MtPf_R, MtPv_F, MtPv_R, MtPm_F, MtPm_R, MtPo_F, MtPo_R, MtPk_F, and MtPt_F were performed. . Specifically, denaturation at 95 ° C. for 15 seconds, annealing at 60 ° C. for 30 seconds, and extension at 72 ° C. for 30 seconds were performed for 30 cycles. By this amplification, 5 types of malaria-specific sequences are amplified using the DNA product amplified in step 2 as a template.
  • the nucleic acid chromatography strip includes DNA strands complementary to DNA tags 1, 2, 3, 4, 7, 8 (tag 1 complementary strand, tag 2 Complementary strand, tag 3 complementary strand, tag 4 complementary strand, tag 7 complementary strand, and tag 8 complementary strand) were printed in a substantially straight line in a direction crossing the longitudinal direction of the strip.
  • the forward primer MtPf_F consisting of the base sequence of SEQ ID NO: 1
  • the reverse primer MtPf_R consisting of the base sequence of SEQ ID NO: 2
  • a line to which complementary DNA is attached is observed in blue by the nanolatex.
  • the PCR product obtained by the above multiplex PCR is added to a developing solution for nucleic acid chromatography to prepare a hybridization solution (Table 4), and a nucleic acid chromatography strip (C-PAS8 (2 mm)) (TBA The lower end of KK) was immersed in the solution, and the PCR product was developed on a nucleic acid chromatography strip at room temperature.
  • FIG. 6 shows the state of the PCR product in the strip 10 minutes after development. Circles indicate the position of the detected band, and genus-specific bands are observed in all five lanes except the negative control (malaria common, Pos). Bands specific for salmalaria in each of the five lanes (Pk), band specific to P. falciparum (Pm), band specific to P. falciparum (Pf), band specific to P. falciparum (Pv), band specific to oval malaria (Po) was observed.
  • nucleic acid chromatography enables rapid detection with a detection time of about 10 minutes after PCR.
  • the present inventors have confirmed that it takes about 3 hours to detect five types of malaria by applying the method described in Non-Patent Document 1, but according to the present invention, this half Five kinds of malaria can be detected in the following time. Further, in the method described in Non-Patent Document 1, it is necessary to use expensive equipment such as an electrophoresis apparatus or a gel photographing apparatus in order to specify the presence or absence of malaria infection and the infected species after PCR. It is not necessary to use this equipment, and significant cost reduction can be expected.
  • expensive equipment such as an electrophoresis apparatus or a gel photographing apparatus

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Abstract

Cette invention concerne un procédé de détection permettant de détecter un ou plusieurs types de parasites paludéens, présents dans un échantillon, choisis dans le groupe constitué par Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, et le parasite paludéen simien. Le procédé de détection comprend : (1) une étape d'amplification d'un acide nucléique à l'aide d'un ensemble d'amorces comprenant 5 types d'amorces ; et (2) une étape de détection d'un ou de plusieurs types de parasites paludéens choisis dans le groupe constitué par Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, et le parasite paludéen simien, sur la base de l'acide nucléique amplifié à l'étape (1).
PCT/JP2018/013219 2017-03-31 2018-03-29 Procédé de détection multiplex pour 5 types de parasites paludéens WO2018181703A1 (fr)

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CN114540526B (zh) * 2022-03-23 2023-10-31 福建医科大学孟超肝胆医院(福州市传染病医院) 对五种输入性疟原虫分型检测的引物、探针及方法

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