WO2018181703A1 - Multiplex detection method for 5 types of malarial parasite - Google Patents

Multiplex detection method for 5 types of malarial parasite Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nucleotide sequence
oligonucleotide
sequence
oligonucleotide represented
Prior art date
Application number
PCT/JP2018/013219
Other languages
French (fr)
Japanese (ja)
Inventor
真弘 平塚
雄大 齋藤
Original Assignee
国立大学法人東北大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人東北大学 filed Critical 国立大学法人東北大学
Publication of WO2018181703A1 publication Critical patent/WO2018181703A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Landscapes

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

Abstract

This detection method is for detecting one or more types of malarial parasites, present in a sample, from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and monkey malarial parasite. The detection method comprises: (1) a step for amplifying a nucleic acid using a primer set comprising 5 types of primers; and (2) a step for detecting one or more types of malarial parasites selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and monkey malarial parasite, on the basis of the nucleic acid amplified in step (1).

Description

５種マラリア原虫のマルチプレックス検出法Multiplex detection method for five species of Plasmodium
　本発明は、5種のマラリア原虫の検出方法、マラリア原虫検出用プライマーセット、及びマラリア原虫検出用キットに関する。 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.
　現在、マラリアは世界で年間およそ2億1200万人の患者が罹患し、死亡者数は42万9000人と報告されている。死亡患者の70%は5歳児未満であり、マラリアと貧困が相互に因果関係となる負のサイクルは、アフリカ等の熱帯地域における社会経済開発を妨げる大きな要因とされる。ヒトに感染する代表的なマラリア原虫には、熱帯熱マラリア（Plasmodium falciparum）、三日熱マラリア（Plasmodium　vivax）、卵形マラリア（Plasmodium ovale）、四日熱マラリア（Plasmodium malariae）及びサルマラリア（Plasmodium knowlesi）の5種類が知られている。マラリア感染の診断の遅れは、治療の遅れに繋がり、重症化の後に死亡する例も少なくない。したがって、マラリア流行地で実施可能な、簡便、迅速、高感度かつ種特異的なマラリア診断法の開発が国際的に喫緊の課題となっている。 Currently, it is reported that malaria affects approximately 212 million patients annually worldwide and the death toll is 429,000. 70% of those who die are under the age of five, and the negative cycle in which malaria and poverty are causally linked is considered to be a major factor impeding socio-economic development in tropical regions such as Africa. Representative 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.
　マラリア原虫の検査は、血液塗抹標本をギムザ染色して光学顕微鏡で観察する方法がゴールドスタンダードである。この顕微鏡法は、熟練者によって確定診断あるいは否定が可能であるが、原虫密度が少ない場合は、長い検査時間と高度な技術が要求され、結果的に治療が遅れる可能性が高い。そのため、イムノクロマトグラフィーを利用したマラリア迅速診断法（Rapid Diagnostic Test; RDT）が開発されている。しかしながら、マラリア種を区別する特異性は低く、感染回復後の偽陽性が問題となっている。 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.
　そこで最近では、ＰＣＲ法を利用したマラリア診断法が開発されている。ＰＣＲ法は顕微鏡法やRDTに比較して感度が高く、混合感染に対しても特異性良く診断できる。しかしながら、最終的なＤＮＡ増幅物の検出をするためには、高額な蛍光検出器の使用や煩雑なゲル電気泳動を行う必要があり、一般病院やマラリア流行地域の現場診療所におけるルーチンな診断には適していない。 Therefore, recently, a malaria diagnostic method using the PCR method has been developed. PCR is more sensitive than microscopy and RDT, and can be diagnosed with high specificity against mixed infections. However, 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.
　非特許文献１には、マラリア原虫のミトコンドリアチトクロームｃオキシダーゼIII（ｃｏｘ３）をターゲットとしたＰＣＲによる、熱帯熱マラリア、三日熱マラリア、卵形マラリア、及び四日熱マラリアの4種類のマラリア原虫のＰＣＲによる検出方法が記載されている。しかしながら、この文献ではサルマラリアの検出が含まれていない。 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.
　従来、三日熱マラリア原虫とサルマラリア原虫のミトコンドリアＤＮＡの相同性は９７％程度と極めて高いことが知られており、一般病院やマラリア流行地域の現場診療で２つの原虫への感染を簡便かつ迅速に識別することは困難である。 Conventionally, the homology of mitochondrial DNA between Plasmodium falciparum and Salmonaria parasites is known to be extremely high at about 97%, and it is easy and easy to infect two protozoa at general hospitals and on-site medical care in malaria endemic areas. It is difficult to identify quickly.
  本発明の目的は、熱帯熱マラリア（Plasmodium falciparum）、三日熱マラリア（Plasmodium vivax）、卵形マラリア（Plasmodium ovale)、四日熱マラリア（Plasmodium malariae）及びサルマラリア（Plasmodium knowlesi）の5種類のマラリア原虫の簡便かつ迅速な検出方法を提供することにある。 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.
　本発明者らは、かかる課題を解決すべく、マラリア原虫のミトコンドリアチトクロームｃオキシダーゼIII（ｃｏｘ３）の塩基配列に基づいて、熱帯熱マラリア、三日熱マラリア、卵形マラリア、四日熱マラリア及びサルマラリアの5種のマラリア原虫の各々を特異的に識別できる２種類以上のオリゴヌクレオチドプライマーを作成し、かかるプライマーからなるプライマーセットにより5種のマラリア原虫に特徴的なＤＮＡ配列をＰＣＲ増幅し、それらのＰＣＲ産物を、当該ＤＮＡ配列に相補的なＤＮＡが取り付けられた核酸検出用デバイスに適用することで、検出されたバンドの位置からマラリア感染の有無及び感染マラリアの原虫種を目視で検出、識別、又は診断できることを見出した。 In order to solve such problems, 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. By applying this PCR product to a nucleic acid detection device to which DNA complementary to the DNA sequence is attached, 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.
　本発明の以下の態様が提供される。 The following aspects of the present invention are provided.
　項１．試料中に存在する、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上の検出方法であって、
　（１）下記の（ａ）～（ｅ）の５種類のプライマーセットを用いて、核酸を増幅する工程、及び
　（２）工程（１）で増幅された核酸に基づいて、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上を検出する工程を含む方法。 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）配列番号１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチド又は配列番号２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
　（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
　（ｃ）配列番号５の塩基配列で表わされるオリゴヌクレオチド又は配列番号５の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチド又は配列番号６の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
　（ｄ）配列番号７の塩基配列で表わされるオリゴヌクレオチド又は配列番号７の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチド又は配列番号８の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、及び
　（ｅ）配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット。 (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 oligonucleotide represented by a nucleotide sequence in which one or two bases have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3; An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P. falciparum, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence of SEQ ID NO: 4 lacking one or two bases 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. falciparum, and 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 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 nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 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 an oval malaria parasite, and (e) SEQ ID NO: 9 1 in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 9 Or an oligonucleotide represented by a nucleotide sequence in which 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 Salmonaria parasite, An oligonucleotide represented by a nucleotide sequence 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: 10, and the cox3 gene sequence of the protozoan parasite A primer set comprising an oligonucleotide for obtaining an amplification product of a specific region.
　項２．工程（１）において、
　（ｆ）配列番号１１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又は配列番号１２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
からなるプライマーセットを用いて、プラスモジウム属の核酸を属特異的に増幅し、
　工程（２）において、工程（１）で増幅された核酸に基づいてプラスモジウム属を検出することをさらに含む項１に記載の方法。 Item 2. In step (1),
(F) 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 Alternatively, 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 Amplified to
Item 2. The method according to Item 1, further comprising detecting Plasmodium based on the nucleic acid amplified in Step (1) in Step (2).
　項３．工程（１）は、
　（ｇ）配列番号１３の塩基配列で表わされるオリゴヌクレオチド又は配列番号１３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１４の塩基配列で表わされるオリゴヌクレオチド又は配列番号１４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
からなるプライマーセットを用いて、前記検体から取得したＤＮＡを鋳型として、１次増幅産物を得る工程と、
　前記（ａ）～（ｅ）であるプライマーセット、又は（ｆ）が存在する場合には（ａ）～（ｆ）であるプライマーセットを用いて、前記１次増幅産物を鋳型として、２次増幅産物を得る工程とを含む
項１又は２に記載の方法。 Item 3. Step (1)
(G) 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. Obtaining a primary amplification product,
When the primer set (a) to (e) or (f) is present, the primer set (a) to (f) is used and the primary amplification product as a template for secondary amplification. Item 3. The method according to Item 1 or 2, comprising a step of obtaining a product.
　項４．前記１次増幅産物を得る工程及び前記２次増幅産物を得る工程を１回の反応で行う、項３に記載の方法。 Item 4. 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.
　項５．前記工程（１）が、前記（ａ）～（ｅ）であるプライマーセット、（ｆ）が存在する場合には（ａ）～（ｆ）であるプライマーセット、又は（ｇ）が存在する場合には（ａ）～（ｅ）及び（ｇ）であるプライマーセット若しくは（ａ）～（ｇ）であるプライマーセットを一つの反応液中に混合した状態で増幅することを含む項１～４のいずれか一項に記載の方法。 Item 5. In the 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.
　項６．前記（ａ）～（ｅ）のプライマーセットのそれぞれ、又は（ｆ）が存在する場合には（ａ）～（ｆ）のプライマーセットのそれぞれは、一方のオリゴヌクレオチドにタグ配列が取り付けられ、他方のオリゴヌクレオチドに標識物質又は標識物質導入材料が取り付けられている項１～５のいずれか一項に記載の方法。 Item 6. 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. The method according to any one of Items 1 to 5, wherein a labeling substance or a labeling substance introduction material is attached to the oligonucleotide.
　項７．配列番号１の塩基配列で表わされるオリゴヌクレオチドと配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた熱帯熱マラリア原虫の検出方法。 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.
　項８．配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いた三日熱マラリア原虫の検出方法。 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. malaria 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 Alternatively, 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.
　項９．配列番号５の塩基配列で表わされるオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた四日熱マラリア原虫の検出方法。 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.
　項１０．配列番号７の塩基配列で表わされるオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた卵形マラリア原虫の検出方法。 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.
　項１１．配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いたサルマラリア原虫の検出方法。 Item 11. 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 have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 9; 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.
　項１２．配列番号１の塩基配列で表わされるオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなる熱帯熱マラリア原虫検出用プライマーセット。 Item 12. 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.
　項１３．配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなる三日熱マラリア原虫検出用プライマーセット。 Item 13. 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. malaria 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 Alternatively, 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.
　項１４．配列番号５の塩基配列で表わされるオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチドとからなる四日熱マラリア原虫検出用プライマーセット。 Item 14. 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.
　項１５．配列番号７の塩基配列で表わされるオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチドとからなる卵形マラリア原虫検出用プライマーセット。 Item 15. 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.
　項１６．配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるサルマラリア原虫検出用プライマーセット。 Item 16. 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 have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 9; 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 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.
　項１７．一方のオリゴヌクレオチドにタグ配列が取り付けられ、他方のオリゴヌクレオチドに標識物質又は標識物質導入材料が取り付けられている項１２～１６のいずれか一項に記載のプライマーセット。 Item 17. 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.
　項１８．下記（ａ）～（ｅ）の５種類のプライマーセット、ＤＮＡポリメラーゼ、及びｄＮＴＰを含む、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上の検出用キット。 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）配列番号１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１の塩基配列において１個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチド又は配列番号２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
　（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
　（ｃ）配列番号５の塩基配列で表わされるオリゴヌクレオチド又は配列番号５の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチド又は配列番号６の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
　（ｄ）配列番号７の塩基配列で表わされるオリゴヌクレオチド又は配列番号７の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチド又は配列番号８の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、及び
　（ｅ）配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット。 (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: 3 or an oligonucleotide represented by a nucleotide sequence in which one or two bases have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3; An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P. falciparum, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence of SEQ ID NO: 4 lacking one or two bases 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. falciparum, and 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 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 nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 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 an oval malaria parasite, and (e) SEQ ID NO: 9 1 in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 9 Or an oligonucleotide represented by a nucleotide sequence in which 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 Salmonaria parasite, An oligonucleotide represented by a nucleotide sequence 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: 10, and the cox3 gene sequence of the protozoan parasite A primer set comprising an oligonucleotide for obtaining an amplification product of a specific region.
　項１９．（ｆ）配列番号１１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又は配列番号１２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
からなるプライマーセットをさらに含む項１８に記載の検出用キット。 Item 19. (F) 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 Item 19. The detection kit according to Item 18, further comprising 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.
　項２０．（ｇ）配列番号１３の塩基配列で表わされるオリゴヌクレオチド又は配列番号１３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１４の塩基配列で表わされるオリゴヌクレオチド又は配列番号１４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
からなるプライマーセットをさらに含む、項１８又は１９に記載の検出用キット。 Item 20. (G) 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 Item 20. The detection according to Item 18 or 19, further comprising 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 For kit.
　項２１．前記（ａ）～（ｅ）であるプライマーセット、（ｆ）が存在する場合には（ａ）～（ｆ）であるプライマーセット、又は（ｇ）が存在する場合には（ａ）～（ｅ）及び（ｇ）であるプライマーセット若しくは（ａ）～（ｇ）であるプライマーセットを一つの反応液中に混合した状態で、前記（ａ）～（ｅ）のプライマーセット、（ａ）～（ｆ）のプライマーセット、（ａ）～（ｅ）及び（ｇ）のプライマーセット、又は（ａ）～（ｇ）のプライマーセットを用いた増幅を行う項１８～２０に記載の検出用キット。 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. The detection kit according to Item 18 to 20, wherein amplification is performed using the primer set of f), the primer set of (a) to (e) and (g), or the primer set of (a) to (g).
　項２２．前記（ａ）～（ｅ）のプライマーセットのそれぞれ、又は（ｆ）が存在する場合には（ａ）～（ｆ）のプライマーセットのそれぞれは、一方のオリゴヌクレオチドにタグ配列が取り付けられ、他方のオリゴヌクレオチドに標識物質又は標識物質導入材料が取り付けられている項１８～２１のいずれか一項に記載の検出用キット。 Item 22. 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. The detection kit according to any one of Items 18 to 21, wherein a labeling substance or a labeling substance introduction material is attached to the oligonucleotide.
　本発明によれば、配列番号１～１０の塩基配列で表わされるオリゴヌクレオチド又はその１個又は２個の塩基が修飾された塩基配列で表わされるオリゴヌクレオチドからなるプライマーセットを使用して、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫の５種のヒトマラリア原虫の有無及び種別を簡便かつ迅速に検出することができる。 According to the present invention, 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.
（Ａ）核酸検出用デバイスの一実施形態の平面図、（Ｂ）側面図。(A) The top view of one Embodiment of the device for nucleic acid detection, (B) Side view. 図１の核酸検出用デバイスを試験溶液に浸漬した状態を示す略図。2 is a schematic diagram showing a state in which the nucleic acid detection device of FIG. 1 is immersed in a test solution. 核酸検出用デバイスを透明な外装体により被覆した状態を示す略図。Schematic which shows the state which covered the device for nucleic acid detection with the transparent exterior body. プライマーセットを用いたマラリア種特異的ＰＣＲの略図。Schematic of malaria species specific PCR using primer set. 核酸クロマトグラフィーによるマラリア検出を説明するための概略図。Schematic for demonstrating malaria detection by nucleic acid chromatography. 核酸クロマトグラフィーによる室温でのＰＣＲ産物の展開10分後のストリップにおけるバンドの検出を示す写真。丸印は検出されたバンドの位置を指す。ＮＣはマラリア非感染のヒトＤＮＡを鋳型にＰＣＲ増幅した産物を用いた陰性対照を示す。The photograph which shows the detection of the band in the strip 10 minutes after expansion | deployment of the PCR product at room temperature by nucleic acid chromatography. A circle indicates the position of the detected band. NC represents a negative control using a product obtained by PCR amplification using malaria-uninfected human DNA as a template.
　以下、本発明の好ましい実施形態について説明する。 Hereinafter, preferred embodiments of the present invention will be described.
　本発明の検出方法は、試料中に存在する、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上の検出方法であって、（１）下記の（ａ）～（ｅ）の５種類のプライマーセットを用いて、核酸を増幅する工程、及び（２）工程（１）で増幅された核酸に基づいて、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上を検出する工程を含む。 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）配列番号１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチド又は配列番号２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｃ）配列番号５の塩基配列で表わされるオリゴヌクレオチド又は配列番号５の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチド又は配列番号６の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｄ）配列番号７の塩基配列で表わされるオリゴヌクレオチド又は配列番号７の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチド又は配列番号８の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｅ）配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　なお、本明細書で使用する「試料」とは、マラリア原虫種である熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫特定の５種を含む可能性があり、本発明の検出方法の対象となるヒトから採取した試料であり、特には血液試料、血液試料から精製したＤＮＡ含有試料等を指す。 (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 One in the oligonucleotide represented by the sequence or the nucleotide sequence of SEQ ID NO: 3 Is an oligonucleotide represented by a nucleotide sequence in which two bases have been deleted, inserted or substituted, and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum, SEQ ID NO: An oligonucleotide represented by a base sequence of 4 or an oligonucleotide represented by a base sequence in which one or two bases have been deleted, inserted or substituted in the base sequence of SEQ ID NO: 4, and a primer set comprising an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence (c) an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 or one or two bases in the nucleotide sequence of SEQ ID NO: 5 An oligonucleotide represented by a deleted, inserted or substituted nucleotide sequence, and four An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P. falciparum, and 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 (D) 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. 8 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: 8, and a primer set consisting of an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence (e) one or two bases in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 9 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 Salmonaria parasite, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 10 1 or 2 bases in the nucleotide or the nucleotide sequence of SEQ ID NO: 10 Is a primer set consisting of 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 Salmonaria parasite The “sample” to be used may include five types of Plasmodium falciparum species, Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, oval malaria parasite, and Salmonaria spp. A sample collected from a human subject to the detection method of the present invention, particularly a blood sample, a DNA-containing sample purified from the blood sample, and the like.
　上記の本発明の検出方法で使用する上記の（ａ）～（ｅ）のプライマーセットにおける各オリゴヌクレオチドは、マラリア原虫のミトコンドリアＤＮＡのチトクロームｃオキシダーゼ３遺伝子（ｃｏｘ３）をターゲットとしている。上記の（ａ）～（ｅ）の各プライマーセットの対の一方の各オリゴヌクレオチドは、各マラリア原虫のｃｏｘ３遺伝子の塩基配列に相補的な配列を有し、各プライマーセットは対応するマラリア原虫のｃｏｘ３遺伝子の特定領域を増幅するように作製されている。このため、従来、マラリア原虫のＤＮＡのＰＣＲ検出において長い間標準と考えられてきた１８ｒＤＮＡの配列に基づくＰＣＲに比べて、高感度かつ高効率に５種類のマラリア原虫を検出することができる。 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.
　また、上記の（ａ）～（ｅ）のオリゴヌクレオチドは、５種類のマラリア原虫の一つを各々特異的に検出できるよう、かつクロスリアクションが起こらないように設計されているため、ＰＣＲに用いるプライマーセットをすべて一つの反応液中に混合してマルチプレックスで増幅及び検出することができ、１．５時間以内、好ましくは１時間以内で迅速にマラリア感染の有無及び感染種の特定又は診断を行うことができる。 In addition, the 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.
　また、後述するように（ａ）～（ｆ）の各プライマーセットにおける対となる２つのオリゴヌクレオチドの一方又は両方に、肉眼で観察できる標識物質、又は標識物質導入材料で標識した場合には、ＰＣＲに用いるプライマーセットをすべて一つの反応液中に混合してＰＣＲを行い、増幅産物をストリップ上に展開することで、１本のストリップ上で５種類のマラリア原虫の感染の有無を目視で特定することができる。 Further, as described later, when one or both of the two oligonucleotides that are paired in each of the primer sets (a) to (f) are labeled with a labeling substance that can be observed with the naked eye, or a labeling substance-introducing material, 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.
　核酸クロマトグラフィーストリップを用いた方法は、常温でも行えるため、従来法のようにアガロースゲルの用時調製や低温保存する必要がない。また、高額な蛍光検出器付きサーマルサイクラーも必要ないため、簡便かつ低コストで5種のマラリア原虫を一度に検出できる。 Since 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. In addition, since 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.
　上記の本発明の検出方法で使用する上記の（ａ）～（ｅ）のプライマーセットにおける各オリゴヌクレオチドには、配列番号１～１０の各塩基配列で表わされるオリゴヌクレオチドに加えて、配列番号１～１０の各塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ5種のマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドも包含される。配列番号１～１０の各塩基配列が、増幅産物を得るためプライマーとして機能する限りにおいて、１個又は２個の塩基が欠失、挿入若しくは置換していてもよい。例えば、配列番号１、配列番号２、配列番号５、配列番号６、配列番号７、配列番号８、配列番号１１、配列番号１２の５’端から１番目の塩基が欠失していても、欠失していない場合と比較して感度は劣るものの、各マラリア原虫の属特異的検出は可能であることが判明している。また、１塩基の置換では通常塩基配列とその相補的な配列との対合にほぼ影響がないことも当該技術分野では良く知られている。 In addition to the oligonucleotides represented by the respective base sequences of SEQ ID NOs: 1 to 10, 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. 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.
　一実施形態では、上記工程（１）において、
　（ｆ）配列番号１１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又は配列番号１２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
を用いて、プラスモジウム属の核酸を属特異的に増幅し、
　上記工程（２）において、工程（１）で増幅された核酸に基づいてプラスモジウム属を検出することをさらに含む。 In one embodiment, in the step (1),
(F) 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 Alternatively, 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 Amplified to
The step (2) further includes detecting Plasmodium based on the nucleic acid amplified in the step (1).
　この場合、（ｆ）のプライマーセットを（ａ）～（ｅ）のプライマーセットと同じ反応溶液に入れることで、試料中の５種類のマラリア原虫の種特異的な検出と、プラスモジウム属の属特異的な検出を同時に行うことができる。 In this case, by placing the primer set of (f) in the same reaction solution as the primer sets of (a) to (e), species-specific detection of five types of malaria parasites in the sample and genus-specificity of Plasmodium Detection can be performed simultaneously.
　上記の（ａ）～（ｅ）及び（ｆ）のプライマーセットを用いてＰＣＲを行う場合、　上記の（ａ）～（ｆ）の各プライマーセットにおける対となる２つのオリゴヌクレオチドの一方の５’端には、スペーサーを介して又は介さずに、タグ配列を取り付けてもよい。例えば、（ａ）の配列番号１の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｃ）の配列番号５の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｄ）の配列番号７の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｅ）の配列番号９の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、及び（ｆ）の配列番号１１の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列の各々の５’端にタグ配列が取り付けられてもよい。 When PCR is performed using the primer sets (a) to (e) and (f) above, 5 ′ of one of the two oligonucleotides to be paired in each of the primer sets (a) to (f) above A tag sequence may be attached to the end with or without a spacer. For example, (a) 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 An oligonucleotide represented by the sequence or a modified sequence thereof, an oligonucleotide represented by the base sequence of SEQ ID NO: 7 in (d) or a modified sequence thereof, and an oligonucleotide represented by the base sequence of SEQ ID NO: 9 in (e) or a modified sequence thereof And 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.
　タグ配列を設けることで、複数の核酸配列を同時に検出するときのクロスリアクションがより効果的に防止される。タグ配列とは、５種のマラリア成虫の標的核酸の配列であるとは無関係にタグ間のクロスリアクションを排除した設計がなされた配列であり、好ましくは、１５塩基以上５０塩基以下、より好ましくは、２０塩基以上２５塩基以下である。そのようなスペーサー及びタグ配列は核酸検出の分野において周知である。タグ配列の導入の詳細については、例えばＷＯ２０１３／０３９２２８号を参照されたい。 By providing a tag sequence, cross reaction when detecting a plurality of nucleic acid sequences at the same time is more effectively prevented. 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. Such spacer and tag sequences are well known in the field of nucleic acid detection. For details of introduction of the tag sequence, refer to, for example, WO2013 / 039228.
　スペーサーは、プライマーとタグ配列の間に挿入される、ポリメラーゼ反応阻害領域からなる遺伝子非発現部分である。スペーサーとしては、核酸誘導体を含むこともできるし、非核酸誘導体を含むこともできる。核酸誘導体としては、Ｌ型核酸、３-ｄｅｏｘｙ-２-ｈｙｄｒｏｘｙ-ｄＮ、修飾塩基核酸、損傷塩基核酸、リン酸結合部位修飾核酸、ＲＮＡ、２’-ＯＭｅ-Ｎ、及び、それらの誘導体からなる群から選択される少なくとも１つが挙げられる。非核酸誘導体としては、炭素鎖（Ｃｎ）、ペグ鎖（（ＣＨ２ＣＨ２Ｏ）ｎ）、ジスルフィド含有鎖（ＣｎＳＳＣｎ）、及びジチオールフォスフォロアミダイトからなる群から選択される少なくとも１つが挙げられる。 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.
　また、上記の（ａ）～（ｅ）及び（ｆ）のプライマーセットを用いてＰＣＲを行う場合、ＰＣＲでの標的核酸の増幅後の検出を容易にするために、上記の（ａ）～（ｆ）の各プライマーセットにおける対となる２つのオリゴヌクレオチドの他方、又は２つのオリゴヌクレオチドの両方に、肉眼で観察できる標識物質、又は標識物質導入材料で標識してもよい。例えば、（ａ）の配列番号２の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｂ）配列番号４の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｃ）の配列番号６の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｄ）の配列番号８の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、（ｅ）の配列番号１０の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列、及び（ｆ）の配列番号１２の塩基配列で表わされるオリゴヌクレオチド又はその修飾配列の各々の５’端に標識物質又は標識物質導入材料が取り付けられてもよい。 In addition, when PCR is performed using the primer sets (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. For example, (a) the 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 An oligonucleotide represented by the sequence or a modified sequence thereof, an oligonucleotide represented by the base sequence of SEQ ID NO: 8 in (d) or a modified sequence thereof, an oligonucleotide represented by the base sequence of SEQ ID NO: 10 in (e) or a modified sequence thereof And 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.
　代わりに、配列番号２、配列番号４、配列番号６、配列番号８、配列番号１０、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又はそれらの各々の修飾配列の５’端にタグ配列が取り付けられている場合は、配列番号１、配列番号３、配列番号５、配列番号７、配列番号９、配列番号１１の塩基配列で表わされるオリゴヌクレオチド又はそれらの各々の修飾配列の５’端に標識物質又は標識物質導入材料が取り付けられてもよい。
　標識物質導入材料は、標識物質と結合するための材料であり、例えば、アビジンコートした標識物質と結合するためのビオチンを含み、標識物質の標的核酸への導入を可能にする。 Instead, 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.
　標識物質は、目視（肉眼で）で検出可能な発光又は発色を提示する発光物質又は発色物質であることが好ましい。このような標識物質としては、各種染料、各種顔料、ルミノール、イソルミノール、アクリジニウム化合物、オレフィン、エノールエーテル、エナミン、アリールビニルエーテル、ジオキセン、アリールイミダゾール、ルシゲニン、ルシフェリン及びエクリオンを包含する化学発光物質が挙げられる。さらに、金コロイド若しくはゾル又は銀コロイド若しくはゾルを包含するコロイド若しくはゾル、金属粒子、無機粒子等が挙げられる。 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). Such 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. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols, metal particles, inorganic particles, and the like can be given.
　標識物質は、その一部にラテックス粒子等の粒子を有していてもよい。標識物質の一部を構成するラテックス粒子などの粒子の平均粒子径は、特に限定しないが、例えば、２０ｎｍ以上２０μｍ以下、好ましくは０．１μｍ以上１０μｍ以下、特に好ましくは０．１μｍ以上５μｍ以下、さらに好ましくは０．１５μｍ以上２μｍ以下である。また、固相担体の孔径によって適宜調整される。 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 μm or less, preferably 0.1 μm or more and 10 μm or less, particularly preferably 0.1 μm or more and 5 μm 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.
　好ましくは、粒子は水溶液に懸濁でき、そして水不溶性ポリマー材料からなる粒子である。例えばポリエチレン、ポリスチレン、スチレン－スチレンスルホン酸塩共重合体、アクリル酸ポリマー、メタクリル酸ポリマー、アクリロニトリルポリマー、アクリロニトリル－ブタジエン－スチレン、ポリビニルアセテート－アクリレート、ポリビニルピロリドン又は塩化ビニル－アクリレートが挙げられる。それらの表面上に活性基、例えばカルボキシル、アミノ又はアルデヒド基を有するラテックス粒子も挙げられる。 Preferably, the particles are particles that can be suspended in an aqueous solution and are made of 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.
　標識物質導入材料としては、抗原抗体反応における抗体や、アビジン（ストレプトアビジン）－ビオチンシステムにおけるビオチン、抗ジゴキシゲニン（ＤＩＧ）－ジゴキシゲニン（ＤＩＧ）システムにおけるジゴキシゲニン、又は抗ＦＩＴＣ－ＦＩＴＣシステムにおけるＦＩＴＣ等に代表されるハプテン類などが挙げられる。検出に用いられる標識物質は、上記標識物質導入材料と相互作用する他方の分子又は物質（例えば、抗原、すなわち、ストレプトアビジン、抗ＦＩＴＣなど）であり、オリゴヌクレオチドプローブとのハイブリダイゼーション工程において、あるいはこの工程に先立って、あるいはこの工程後に、増幅産物に取り付けられた標識物質導入材料と、標識物質導入材料と結合する部位を備える標識物質と結合して、増幅産物の検出を可能にする。 Examples of the labeling substance introduction material 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.
　本発明の検出方法の別の実施形態では、上記工程（１）は、（ｇ）配列番号１３の塩基配列で表わされるオリゴヌクレオチド又は配列番号１３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、及び配列番号１４の塩基配列で表わされるオリゴヌクレオチド又は配列番号１４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いて、前記検体から取得したＤＮＡを鋳型として、１次増幅産物を得る工程と、前記（ａ）～（ｅ）のプライマーセット、又は（ｆ）が存在する場合には（ａ）～（ｆ）のプライマーセットを用いて、前記１次増幅産物を鋳型として、２次増幅産物を得る工程とを含む。 In another embodiment of the detection method of the present invention, 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 Alternatively, in order to obtain an amplification product of 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: 14, and a specific region of the Plasmodium cox3 gene sequence Obtained from the specimen using 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.
　このような方法（Nested PCR）によれば、検出感度の向上や増幅時のノイズを回避できるため、５種類のマラリア原虫をより高感度かつ特異的に検出することができる。なお、一般的なNested PCRでは、１つ目のプライマーセットを用いてＰＣＲ反応を行った後（１次増幅）、増幅された遺伝子（増幅産物）の内側の領域を２つ目のプライマーセットを用いて増幅（２次増幅）するため、ＰＣＲ反応を２回行う必要がある。しかしながら、本発明では、１次増幅用プライマーセット（（ｇ）のプライマーセット）と、プラスモジウム属共通のプライマーセット（（ｆ）のプライマーセット）と、種特異的なプライマーセット（（ａ）～（ｅ）のプライマーセット）とのアニーリング温度がそれぞれ異なるように、かつ、種特異的プライマーセット（（ａ）～（ｅ）のプライマーセット）のアニーリング温度が同一となるように設計しているため、反応条件を工夫することで、１回のＰＣＲ反応でNested PCRを達成し、５種類のマラリア原虫を区別して検出することができる。 According to such a method (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. In general 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. However, in the present invention, 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, By devising the reaction conditions, it is possible to achieve Nested PCR in one PCR reaction and to distinguish and detect five types of malaria parasites.
　本発明におけるＰＣＲ条件は、目的のＤＮＡ断片を検出可能な程度に増幅することができれば特に制限されず、例えば、ＰＣＲ反応に用いる鋳型としての２本鎖ＤＮＡ量は０ ．１ｎｇ以上が好ましく、１～５０ｎｇがより好ましい。また、２本鎖ＤＮＡを１本鎖にする熱変性反応を９２～９８℃で１０～６０秒間行い、プライマー対を１本鎖ＤＮＡにハイブリダイズさせるアニーリング反応を５５～７０℃で１０～６０秒間行い、ＤＮＡポリメラーゼを作用させる伸長反応を７１～７５℃で５～１２０秒間行い、これらを１サイクルとしたものを２０～４０サイクル行う。サイクル反応の前に初期変性反応を行うことが好ましい。ＰＣＲにおける初期変性反応条件について、変性温度は９２～９８℃が好ましく、変性時間は３０秒～１０分が好ましい。なお、上述したように、１回の反応でNested PCRを行う場合、１次増幅は、熱変性反応を９２～９８℃で１０～６０秒間行い、アニーリング反応を６５～７０℃で１０～６０秒間行い、伸長反応を７１～７５℃で１０～６０秒間行い、これらを１サイクルとしたものを１０～３０サイクル行うことが好ましく、２次増幅は、熱変性反応を９２～９８℃で１０～６０秒間行い、アニーリング反応を１次反応のアニーリング温度よりも低い温度、例えば５５～６５℃で１０～６０秒間行い、伸長反応を７１～７５℃で１０～６０秒間行い、これらを１サイクルとしたものを２０～４０サイクル行うことが好ましい。 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. For example, the amount of double-stranded DNA as a template used in the PCR reaction is 0 μm. 1 ng or more is preferable, and 1 to 50 ng is more preferable. In addition, 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. for 5 to 120 seconds, and these are defined as one cycle for 20 to 40 cycles. It is preferable to perform an initial denaturation reaction before the cycle reaction. Regarding the initial denaturation reaction conditions in PCR, the denaturation temperature is preferably 92 to 98 ° C., and the denaturation time is preferably 30 seconds to 10 minutes. As described above, when performing Nested PCR in one reaction, 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. In the secondary amplification, the heat denaturation reaction is carried out at 92 to 98 ° C. for 10 to 60 cycles. For 1 second, and 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.
　本発明は、配列番号１の塩基配列で表わされるオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた熱帯熱マラリア原虫の検出方法を包含する。
　試料中の核酸を増幅する工程及び増幅された核酸に基づいて熱帯熱マラリア原虫を検出する工程については、上記の工程（１）及び工程（２）で説明した通りである。
　この方法によれば、熱帯熱マラリア原虫を種特異的に検出することができる。 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.
　本発明は、配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いた三日熱マラリア原虫の検出方法を包含する。
　試料中の核酸を増幅する工程及び増幅された核酸に基づいて三日熱マラリア原虫を検出する工程については、上記の工程（１）及び工程（２）で説明した通りである。 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 of amplifying the nucleic acid in the sample and the step of detecting Plasmodium vivax based on the amplified nucleic acid are as described in the above step (1) and step (2).
　この方法によれば、三日熱マラリア原虫を種特異的に検出することができる。 According to this method, Plasmodium falciparum can be detected species-specifically.
　本発明は、配列番号５の塩基配列で表わされるオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた四日熱マラリア原虫の検出方法を包含する。
　試料中の核酸を増幅する工程及び増幅された核酸に基づいて四日熱マラリア原虫を検出する工程については、上記の工程（１）及び工程（２）で説明した通りである。 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).
　この方法によれば、四日熱マラリア原虫を種特異的に検出することができる。 According to this method, Plasmodium falciparum can be detected species-specifically.
　本発明は、配列番号７の塩基配列で表わされるオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた卵形マラリア原虫の検出方法を包含する。
　試料中の核酸を増幅する工程及び増幅された核酸に基づいて卵形マラリア原虫を検出する工程については、上記の工程（１）及び工程（２）で説明した通りである。 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).
　この方法によれば、卵形マラリア原虫を種特異的に検出することができる。 According to this method, the oval malaria parasite can be detected in a species-specific manner.
　本発明は、配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いたサルマラリア原虫の検出方法を包含する。
　試料中の核酸を増幅する工程及び増幅された核酸に基づいてサルマラリア原虫を検出する工程については、上記の工程（１）及び工程（２）で説明した通りである。 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 A method for detecting a protozoan malaria using 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 the protozoa Is included.
The step of amplifying the nucleic acid in the sample and the step of detecting the protozoan protozoa based on the amplified nucleic acid are as described in the above step (1) and step (2).
　この方法によれば、サルマラリア原虫を種特異的に検出することができる。 According to this method, plasmodium parasite can be detected in a species-specific manner.
　本発明は、配列番号１の塩基配列で表わされるオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなる熱帯熱マラリア原虫検出用プライマーセットを包含する。 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.
　かかるプライマーセットは熱帯熱マラリア原虫検出用の特異的プライマーセットとして使用することができる。 Such a primer set can be used as a specific primer set for detecting Plasmodium falciparum.
　本発明は、配列番号１の塩基配列で表わされるオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなる熱帯熱マラリア原虫検出用プライマーセットを包含する。 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.
　本発明は、配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなる三日熱マラリア原虫検出用プライマーセットを包含する。 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.
　かかるプライマーセットは三日熱マラリア原虫検出用の特異的プライマーセットとして使用することができる。 Such a primer set can be used as a specific primer set for detecting Plasmodium falciparum.
　本発明は、配列番号５の塩基配列で表わされるオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチドとからなる四日熱マラリア原虫検出用プライマーセットを包含する。 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.
　かかるプライマーセットは四日熱マラリア原虫検出用の特異的プライマーセットとして使用することができる。 Such a primer set can be used as a specific primer set for detecting Plasmodium falciparum.
　本発明は、配列番号７の塩基配列で表わされるオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチドとからなる卵形マラリア原虫検出用プライマーセットを包含する。 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.
　かかるプライマーセットは卵形マラリア原虫検出用の特異的プライマーセットとして使用することができる。 Such a primer set can be used as a specific primer set for detecting oval malaria parasites.
　本発明は、配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるサルマラリア原虫検出用プライマーセットを包含する。 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.
　かかるプライマーセットはサルマラリア原虫検出用の特異的プライマーセットとして使用することができる。 Such a primer set can be used as a specific primer set for detecting Plasmodium vivax.
　さらに、配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなる第１のプライマーセットと、配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなる第２プライマーセットとを用いた三日熱マラリア原虫とサルマラリア原虫の識別方法を包含する。 Furthermore, 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 A first 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, SEQ ID NO: In the oligonucleotide represented by the nucleotide sequence of 9 or the nucleotide sequence of SEQ ID NO: 9 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 Salmonaria parasite, SEQ ID NO: An oligonucleotide represented by 10 nucleotide sequences 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: 10, and the cox3 gene of Salmonaria parasite It includes a method for distinguishing Plasmodium vivax and Plasmodium falciparum using a second primer set comprising an oligonucleotide for obtaining an amplification product of a specific region of the sequence.
　上記のプライマーセットは、ＰＣＲ法に用いてもよいし、一定温度等温で核酸を増幅する遺伝子増幅法であるＬＡＭＰ法や、ＳＤＡ法、ＩＣＡＮ法、ＲＣＡ法に用いてもよい。 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.
　本発明はさらに、下記（ａ）～（ｅ）の５種類のプライマーセット、ＤＮＡポリメラーゼ、ｄＮＴＰ、及び反応緩衝液を含む、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上の検出用キットを包含する。 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）配列番号１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１の塩基配列において１個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチド又は配列番号２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｃ）配列番号５の塩基配列で表わされるオリゴヌクレオチド又は配列番号５の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチド又は配列番号６の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｄ）配列番号７の塩基配列で表わされるオリゴヌクレオチド又は配列番号７の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチド又は配列番号８の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット
　（ｅ）配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット (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 oligonucleotide sequence or the nucleotide sequence of SEQ ID NO: 3 An oligonucleotide having a nucleotide sequence deleted, inserted or substituted, and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Plasmodium falciparum, and the nucleotide sequence of SEQ ID NO: 4 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: 4, and the cox3 gene sequence of Plasmodium vivax Primer set consisting of an oligonucleotide for obtaining an amplification product of a specific region (c) One or two bases deleted or inserted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 or the nucleotide sequence of SEQ ID NO: 5 Or 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 the protozoan parasite, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence of SEQ ID NO: 6 with one or two bases deleted; A primer set comprising an oligonucleotide represented by an 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 (d) a base of SEQ ID NO: 7 An oligonucleotide represented by a sequence 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: 7, and the cox3 gene sequence of an oval malaria parasite An oligonucleotide for obtaining an amplification product of a specific region, and a salt of SEQ ID NO: 8 An oligonucleotide represented by a base sequence 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: 8, and the cox3 gene sequence of an oval malaria parasite A primer set comprising an oligonucleotide for obtaining an amplification product of the specific region of (e) one or two bases deleted in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 9, An oligonucleotide represented by the inserted or substituted base sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Salmonaria parasite, and the oligonucleotide or the sequence represented by the base sequence of SEQ ID NO: 10 1 or 2 bases are deleted in the base sequence of number 10. A primer set comprising an oligonucleotide represented by an inserted or substituted nucleotide sequence and an oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of Salmonaria parasite
　ＤＮＡポリメラーゼは、鎖置換活性を有する鋳型依存性核酸合成酵素であればいずれでもよい。当該ポリメラーゼとしては、Bst DNAポリメラーゼ（ラージフラグメント）、Bca(exo-)DN Aポリメラーゼ、大腸菌DNAポリメラーゼＩのクレノウフラグメント、Vent (Exo-)DNAポリメラーゼ（Vent DNAポリメラーゼからエクソヌクレアーゼ活性を除いたもの）、DeepVent (Exo-)DNAポリメラーゼ（DeepVent DNAポリメラーゼからエクソヌクレアーゼ活性を除いたもの）、KOD DNAポリメラーゼなどが挙げられる。 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としては、ｄＡＴＰ、ｄＴＴＰ、ｄＧＴＰ、ｄＣＴＰが挙げられる。 DNTP includes dATP, dTTP, dGTP, and dCTP.
　反応緩衝液としては、トリス緩衝液、ＥＤＴＡ緩衝液、及びトリス－ＥＤＴＡ緩衝液等が挙げられる。 Examples of the reaction buffer include Tris buffer, EDTA buffer, and Tris-EDTA buffer.
　一実施形態において、上記検出用キットは、（ｆ）配列番号１１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又は配列番号１２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットをさらに含む。 In one embodiment, 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.
　この場合、一つのキットで５種類のマラリア原虫の種特異的な検出と同時に、プラスモジウム属の属特異的な検出も同時に行うことができる。
　上記検出用キットは、上記の（ａ）～（ｅ）の５種類のプライマーセット及び任意選択で（ｆ）のプライマーセットを一つの反応液中に混合した状態で増幅及び検出を行うことができ、１．５時間以内、好ましくは１時間以内で迅速にマラリア感染の有無及び感染種の特定又は診断を行うことができる。 In this case, 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. Within 1.5 hours, preferably within 1 hour, the presence or absence of malaria infection and the identification or diagnosis of the infected species can be performed quickly.
　別の実施形態において、上記検出用キットは、（ｇ）配列番号１３の塩基配列で表わされるオリゴヌクレオチド又は配列番号１３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１４の塩基配列で表わされるオリゴヌクレオチド又は配列番号１４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットをさらに含む。 In another embodiment, 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.
　このような検出キットを用いてNaseted PCRを行った場合、検出感度の向上やノイズを回避できるため、５種マラリア原虫をより高感度かつ特異的に検出することができる。なお、上述したように、本発明では、１次増幅用プライマーセット（（ｇ）のプライマーセット）と、プラスモジウム属共通のプライマーセット（（ｆ）のプライマーセット）と、種特異的なプライマーセット（（ａ）～（ｅ）のプライマーセット）とのアニーリング温度がそれぞれ異なるように、かつ、種特異的プライマーセット（（ａ）～（ｅ）のプライマーセット）のアニーリング温度が同一となるように設計しているため、反応条件を工夫することで、１回のＰＣＲ反応でNested PCRを達成し、５種類のマラリア原虫を区別して検出することができる。 When Naseted PCR is performed using such a detection kit, detection sensitivity can be improved and noise can be avoided, so that the five species of Plasmodium can be detected with higher sensitivity and specificity. As described above, in the present invention, 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.
　５種類のマラリア原虫の標的核酸配列は、上記の（ａ）～（ｆ）の各プライマーセットにおける対となる２つのオリゴヌクレオチドの一方に取り付けられたタグ配列とハイブリダイズし得る塩基配列を有するオリゴヌクレオチドプローブを保持する固相担体を備えた核酸検出用デバイスにより、検出することができる。 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.
　図１（Ａ）は核酸検出用デバイスの一実施形態の平面図であり、図１（Ｂ）は同核酸検出用デバイスの側面図である。 FIG. 1 (A) is a plan view of an embodiment of a nucleic acid detection device, and FIG. 1 (B) is a side view of the nucleic acid detection device.
　一実施形態の核酸検出用デバイス１は、固相担体１０を備えている。固相担体１０は、検出対象であるマラリア原虫の標的核酸の検出用のオリゴヌクレオチドプローブを保持する多孔質材料を有する。 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.
　「オリゴヌクレオチドプローブ」は、検出対象であるマラリア原虫の標的核酸の増幅用の上述のオリゴヌクレオチドプライマーに取り付けられたＤＮＡタグにハイブリダイズし得る、該ＤＮＡタグの配列に相補的な配列を有するオリゴヌクレオチドとすることもできるし、マラリア原虫の標的核酸と特異的にハイブリダイズしうる配列を有するように設計されたオリゴヌクレオチドとすることもできる。オリゴヌクレオチドプローブのオリゴヌクレオチド配列の長さは、特に限定されないが、ハイブリダイズする核酸に対する特異性とハイブリダイゼーション効率を確保するため、１５塩基以上５０塩基以下であることが好ましい。 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.
　多孔質材料は当該分野で周知であり、例えば、セルロース、ニトロセルロース、ナイロン等が挙げられる。固相担体１０はストリップ状又はシート状に通常形成される。オリゴヌクレオチドプローブの配列の長さは、特に限定されないが、各標的核酸に対する特異性とハイブリダイゼーション効率を確保するため、１５塩基以上５０塩基以下であることが好ましい。 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.
　任意選択で、核酸検出用デバイス１は固相担体１０を支持するバッキング部材１２を備えてもよい。さらに任意選択で、核酸検出用デバイス１は、固相担体１０と外部環境との間の気体の流通を妨げ、オリゴヌクレオチドプローブと標的核酸とのハイブリダイゼーション反応環境の気密性を維持する被覆部材１４を備えてもよい。被覆部材１４は固相担体の表面を被覆する透明フィルムであり、透明フィルムを構成する素材は、固相担体表面の気密性を維持し、展開液の蒸発を妨げるものであれば特に限定されないが、例えば、ポリエチレンテレフタレートを挙げることができる。被覆部材１４を設けることにより、核酸クロマトグラフィーにおける非特異的シグナル（非特異的ハイブリダイゼーション）の発生を簡便に抑制でき、標的核酸をより高感度に検出することができる。 Optionally, 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.
　図１（Ａ），（Ｂ）において、オリゴヌクレオチド固定領域１６ａ，１６ｂ，１６ｃ，１６ｄ，１６ｅ，１６ｆは、固相担体１０におけるオリゴヌクレオチドプローブの固定位置であり、固相担体１０の先端部から一定程度離れた位置に、対応する標的核酸ごとにライン状に互いに平行に固定される。 1A and 1B, 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.
　オリゴヌクレオチドプローブの固相担体１０への固定化方法は特に限定されず、その３’末端で固相担体１０に結合されていてもよいし、５’末端で結合されていてもよい。例えば、ニトロセルロース等を固相担体１０に使用する場合、プローブを固相担体１０にインクジェット方式でプリントした後、ＵＶ照射などにより固着性を高める。この方法の場合、プローブはＵＶの届く固相担体１０表面のみに固定化される。 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. For example, when nitrocellulose or the like is used for the solid phase carrier 10, after the probe is printed on the solid phase carrier 10 by an ink jet method, the fixing property is enhanced by UV irradiation or the like. In the case of this method, the probe is immobilized only on the surface of the solid-phase carrier 10 where UV reaches.
　固相担体１０上には標的核酸に対応するプローブ領域を容易に特定できるように、１つ又は複数の位置マーカー１８ａ，１８ｂ，１８ｃを配置してもよい。位置マーカー１８ａ，１８ｂ，１８ｃの存在により、目視検出の場合であっても、簡便に標的核酸の存在又は不存在を検出することができる。 On the solid phase carrier 10, 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.
　ここで、ＰＣＲで増幅した試料を用いた核酸検出用デバイス１での核酸検出を例として説明する。核酸検出用デバイス１でのマラリア原虫の標的核酸の検出に先立ち、試料中のＤＮＡからマラリア原虫のｃｏｘ３遺伝子配列の特定領域を、かかる標的核酸を含む試料を第１プライマー及び第２プライマーからなる増幅用プライマーセットを用いて増幅する。 Here, the nucleic acid detection using the nucleic acid detection device 1 using a sample amplified by PCR will be described as an example. Prior to detection of the target nucleic acid of the malaria parasite by the nucleic acid detection device 1, 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.
　第１プライマーは、標的核酸に相補的な、標的核酸中の第１の塩基配列を識別する第１の識別配列とタグ配列とを有するが、タグ配列と第１の識別配列とは直接連結されず、その間にＤＮＡポリメラーゼ反応を抑制又は停止可能な連結部位を有することが好ましい。この連結部位は、天然塩基又は天然塩基と対合する天然塩基の誘導体（天然塩基等）を含まず、例えば、人工オリゴヌクレオチドから構成され、それゆえＤＮＡポリメラーゼ反応を抑制又は停止させる。これにより、一本鎖領域を有する核酸が増幅され、熱変性なしにオリゴヌクレオチドプローブとのハイブリダイゼーションが可能となる。 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. Thereby, a nucleic acid having a single-stranded region is amplified, and hybridization with an oligonucleotide probe becomes possible without thermal denaturation.
　第１プライマーとＰＣＲにおいて対となる第２プライマーは、標的核酸に相補的な、標的核酸中の第２の塩基配列を識別する第２の識別配列に加えて、増幅された標的核酸がオリゴヌクレオチドプローブとハイブリダイズしたときに目視確認可能な標識物質、又は標識物質導入材料を含む。標識物質及び標識物質導入材料については上述した通りである。 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.
　試料中のＤＮＡからマラリア原虫のｃｏｘ３遺伝子配列の特定領域を増幅させた後の増幅産物を含む試験溶液（展開液）を、固相担体１０の先端に適用する。適用された試験溶液は、固相担体１０を構成する多孔質材料内にキャピラリー現象によって展開される。 A test solution (developing 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.
　図２に示すように、固相担体１０の先端を、エッペンドルフチューブ等の容器２０内に入れた増幅された標的核酸を含む試験溶液２２に浸漬させることで固相担体１０に展開することができる。 As shown in FIG. 2, 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. .
　代わりに、図３に示すように、固相担体１０の全体を被覆し、少なくとも一部が透明な外装部材３０の先端部３２付近に固相担体１０の先端を配置し、外装部材３０の先端部３２の先端に設けられたサンプルポート３４を介して容器２０中の試験溶液２２を外装部材３０内に入れて、固相担体１０の先端を浸漬させることでも固相担体１０に展開され得る。外装部材３０は、標的核酸を目視検出できるように、少なくとも一部は透明でなければならない。 Instead, as shown in FIG. 3, 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.
　試験溶液は、固相担体１０中での標的核酸の展開を容易にするように、「展開媒体」を含むことが好ましい。展開媒体は、特に限定されないが、例えば、水、水と相溶する有機溶媒、又は水と１種又は２種以上の前記有機溶媒の混液が挙げられる。水と相溶する有機溶媒としては、例えば、炭素数１～４程度の低級アルコール、ＤＭＳＯ、ＤＭＦ、酢酸メチル、酢酸エチルなどのエステル類、アセトン等が挙げられる。展開媒体は、好ましくは水を主体とする。 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. Examples of 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.
　展開媒体は、ｐＨを調整するための緩衝成分を含むことができる。緩衝成分は、意図するｐＨにもよるが、通常は６．０以上８．０以下の範囲である。より好ましくは、７．０以上８．０以下である。こうしたｐＨを得るための成分は、例えば、酢酸と酢酸ナトリウム（酢酸緩衝液）、クエン酸とクエン酸ナトリウム（クエン酸緩衝液）、リン酸とリン酸ナトリウム（リン酸緩衝液）等である。さらに、リン酸緩衝生理食塩水（ＰＢＳ）等が挙げられる。なお、展開媒体として、増幅反応液をそのまま採用してもよい。また、増幅反応液に対して、界面活性剤や適当な塩等の追加の成分や溶媒を加えたりすることで、組成や濃度の調整をして展開媒体として使用してもよい。展開時間は特に限定されず、固相担体１０の形態や形状、展開媒体の性質に応じて適宜設定される。 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. Furthermore, a phosphate buffered saline (PBS) etc. are mentioned. In addition, you may employ | adopt an amplification reaction liquid as a developing medium as it is. Further, the 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.
　固相担体１０に保持されたオリゴヌクレオチドプローブが、第１プライマーに取り付けられたＤＮＡタグにハイブリダイズし得る該ＤＮＡタグの配列に相補的な配列を有するため、第１プライマーとハイブリダイズし、第１プライマーは標識物質又は標識物質導入材料を含む第２プライマーとハイブリダイズするため、オリゴヌクレオチドプローブを固定化したラインの発色により、複数の標的核酸を種特異的に及び／属特異的に目視で識別して検出することができる。 Since 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.
　以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されない。 Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
１．試料の収集
　大阪市立大学大学院医学研究科の金子明教授のグループが、2015年にマラリア原虫ミトコンドリアDNAのcox3遺伝子をターゲットとして、種特異的検出する方法を報告しており（Isozumi, R., et al., Parasitol Int, 2015）、同法を参考にしてPCR条件を検討した。標準DNAとして、5種のマラリアに単独感染したケニア人及びヴァヌアツ人から採取した血液由来の精製済みDNA（大阪市立大学より提供）を使用した。 1. Sample collection Prof. Akira Kaneko's group at Osaka City University Graduate School of Medicine reported a species-specific detection method targeting the cox3 gene of Plasmodium mitochondrial DNA in 2015 (Isozumi, R., et. al., Parasitol Int, 2015), and PCR conditions were examined with reference to the same method. As standard DNA, purified DNA derived from blood (provided by Osaka City University) collected from Kenyans and Vanuatu who were infected with 5 types of malaria alone was used.
２．マルチプレックスＰＣＲ
　表１に、５種マラリア原虫マルチプレックス検出のためのマルチプレックスＰＣＲで用いたオリゴヌクレオチドプライマーの塩基配列を示す。 2. 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.
　プラスモジウム属の属特異的なプライマーを、Plasmodium のｃｏｘ３遺伝子配列に基づいてParasitology International 64 (2015)304-308を参照して設計した。MtPuni_F（配列番号１１）及びMtPuni_R（配列番号１２）がそれぞれ属特異的な順方向プライマー及び逆方向プライマーである。また、熱帯熱マラリア、三日熱マラリア、卵形マラリア、四日熱マラリア及びサルマラリアの5種の種特異的なプライマーを、各々のマラリア原虫のｃｏｘ３遺伝子配列に基づいて設計した。MtPf_F （配列番号１）及びMtPf_R（配列番号２）がそれぞれ熱帯熱マラリアに特異的な順方向プライマー及び逆方向プライマーであり、MtPv_F（配列番号３）及びMtPv_ R（配列番号４）がそれぞれ三日熱マラリアに特異的な順方向プライマー及び逆方向プライマーであり、MtPm_F（配列番号５）及びMtPm_ R（配列番号６）がそれぞれ四日熱マラリアに特異的な順方向プライマー及び逆方向プライマーであり、MtPo_F（配列番号７）及びMtPo_ R（配列番号８）がそれぞれ卵形マラリアに特異的な順方向プライマー及び逆方向プライマーであり、MtPk_F（配列番号９）及びMtPk_ R（配列番号１０）がそれぞれサルマラリアに特異的な順方向プライマー及び逆方向プライマーである。 A genus-specific primer of the genus Plasmodium was designed with reference to Parasitology International 64 (2015) 304-308 based on the cox3 gene sequence of Plasmodium. MtPuni_F (SEQ ID NO: 11) and MtPuni_R (SEQ ID NO: 12) are gene-specific forward primers and reverse primers, respectively. In addition, 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) and MtPf_R (SEQ ID NO: 2) are forward and reverse primers specific for P. falciparum respectively, and 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_ 番号 R (SEQ ID NO: 6) are respectively a forward primer and a reverse primer specific for P. falciparum malaria, MtPo_F (SEQ ID NO: 7) and MtPo_ R (SEQ ID NO: 8) are forward and reverse primers specific for oval malaria, respectively, and MtPk_F (SEQ ID NO: 9) and MtPk_ R (SEQ ID NO: 10) are monkeys, respectively. Forward and reverse primers specific for malaria.
　各PCRプライマーは、各マラリア種に特徴的なcox3遺伝子配列に続き、スペーサー（3つのカーボン連結）、DNAタグ配列を連結する。図４に示すように、配列番号１，３，５，７，９，１１の塩基配列からなる順方向プライマーの５’末端にはそれぞれＤＮＡタグ１，２，４，３，７，８を付け、それらの各々と対をなす配列番号２，４，６，８，１０，１２の塩基配列からなる逆方向プライマーの５’末端にはビオチンを付加した。 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. As shown in FIG. 4, 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.
　DNAタグ付きプライマー及びビオチン付加プライマーはTBA株式会社で合成した。 Primers with DNA tags and biotin-added primers were synthesized by TBA Corporation.
　また、一次増幅用の順方向プライマーMtU_F（配列番号１）及び逆方向プライマーMtU_R（配列番号２）も設計した。一次増幅用の順方向プライマーMtU_F及び逆方向プライマーMtU_RはFASMAC社で合成した。 In addition, 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. Forward primer MtU_F and reverse primer MtU_R for primary amplification were synthesized by FASMAC.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表２に、ＰＣＲに使用した試薬及びプライマーの量を示す。試薬及びすべてのプライマーを一つの反応液中に混合し、PCR 0.2 ml チューブバンド　セパレート8連　ナチュラル（Greiner Bio-One）に入れ、ＰＣＲに供した。 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.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表３に、ＰＣＲ条件を示す。T100　サーマルサイクラー（Bio-Rad）を使用した。反応温度を３段階にすることで、1本のチューブ内での１回の反応でNested PCRを可能にした。ステップ１として、95°C、3分の変性を1回行った（初期変性反応）。次に、ステップ２として、１次増幅用のプライマーMtU_F 及びMtU_Rによる１次増幅を行った。具体的には、変性95℃，15秒、アニーリング 70°C，30秒、伸長72℃，30秒を10サイクル行った。この増幅により、5種のマラリアの共通配列が増幅される。最後に、ステップ３として、属特異的プライマーMtPuni_F及びMtPuni_R及び５種のマラリア原虫特異的プライマーMtPf_F、MtPf_R、MtPv_F、MtPv_R、MtPm_F、MtPm_R、MtPo_F、MtPo_R、MtPk_F、及びMtPk_Rによる２次増幅を行った。具体的には、変性95℃，15秒、アニーリング 60°C，30秒、伸長72℃，30秒を30サイクル行った。この増幅により、ステップ２で増幅されたDNA産物を鋳型として、5種のマラリアに特異的な配列が増幅される。 Table 3 shows the PCR conditions. A T100 thermal cycler (Bio-Rad) was used. Nested PCR can be performed in one reaction in one tube by setting the reaction temperature to three stages. As step 1, denaturation was performed once at 95 ° C. for 3 minutes (initial denaturation reaction). Next, as step 2, 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. Finally, as step 3, 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.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
３．核酸検出デバイスを用いた核酸クロマトグラフィー
　図５に示すように、核酸クロマトグラフィーストリップには、ＤＮＡタグ１，２，３，４，７，８に相補的なＤＮＡ鎖（タグ１相補鎖、タグ２相補鎖、タグ３相補鎖、タグ４相補鎖、タグ７相補鎖、タグ８相補鎖）を、ストリップの長手方向に対して交差する方向に略直線状にプリントしておいた。このような構成にすることで、例えば配列番号１の塩基配列からなる順方向プライマーMtPf_Fと配列番号２の塩基配列からなる逆方向プライマーMtPf_Rにより増幅されたＤＮＡがある場合には、ＤＮＡタグ１に相補的なＤＮＡが取り付けられたラインがナノラテックスにより青色に呈色して観察される。 3. Nucleic Acid Chromatography Using Nucleic Acid Detection Device As shown in FIG. 5, 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. By adopting such a configuration, for example, when there is DNA amplified by the forward primer MtPf_F consisting of the base sequence of SEQ ID NO: 1 and 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.
　上記のマルチプレックスＰＣＲで得られたＰＣＲ産物を、核酸クロマトグラフィー用の展開液に加えてハイブリダイズ用溶液を調製し（表４）、核酸クロマトグラフィーストリップ（C-PAS8 (2 mm)）（TBA株式会社）の下端を当該溶液に浸漬し、ＰＣＲ産物を室温にて核酸クロマトグラフィーストリップに展開した。 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.
　図６に、展開10分後のストリップにおけるＰＣＲ産物の様子を示す。丸印は検出されたバンドの位置を指し、陰性対照以外の５つのレーンのすべてにおいて属特異的なバンドが観察され（マラリア共通、Pos）、５つのレーンのそれぞれでサルマラリアに特異的なバンド（Pk）、四日熱マラリアに特異的なバンド（Pm）、熱帯熱マラリアに特異的なバンド（Pf）、三日熱マラリアに特異的なバンド（Pv）、卵形マラリアに特異的なバンド（Po）が観察された。 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.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　本実施例では、５種のマラリア原虫を特異的に、かつ、1本のチューブ内で１回のＰＣＲ反応で増幅した上で、ＰＣＲ産物を核酸クロマトグラフィーストリップに展開し、検出されたバンドの位置を肉眼で識別又は診断することで、マラリア感染の有無及び感染マラリアの原虫種を容易に特定できる検出又は診断デバイスを開発した。特に、このデバイスでは、nested PCRとマルチプレックスＰＣＲを組み合わせることにより操作の簡便化と検出の高感度化を可能としている。また、核酸クロマトグラフィーにより、ＰＣＲ後の検出を約１０分とする迅速化を可能としている。 In this example, five kinds of malaria parasites were specifically and amplified by one PCR reaction in one tube, and then the PCR product was developed on a nucleic acid chromatography strip. By detecting or diagnosing the position with the naked eye, we developed a detection or diagnostic device that can easily identify the presence or absence of malaria infection and the protozoan species of infected malaria. In particular, in this device, it is possible to simplify the operation and increase the sensitivity of detection by combining nested PCR and multiplex PCR. In addition, nucleic acid chromatography enables rapid detection with a detection time of about 10 minutes after PCR.
　本発明者らは、非特許文献１に記載の方法を応用して５種マラリアを検出した場合には３時間程度の時間を要することを確認しているが、本発明によれば、この半分以下の時間で５種マラリアの検出が可能となる。また、非特許文献１に記載の方法では、ＰＣＲ後にマラリア感染の有無及び感染種を特定するため、電気泳動装置やゲル撮影装置などの高額な機器を使用する必要があるが、本発明ではこれらの機器を使用する必要はなく、大幅な低コスト化が期待できる。 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.

Claims (22)

  1. 　試料中に存在する、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上の検出方法であって、
    　（１）下記の（ａ）～（ｅ）の５種類のプライマーセットを用いて、核酸を増幅する工程、及び
    　（２）工程（１）で増幅された核酸に基づいて、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上を検出する工程を含む方法。
    　（ａ）配列番号１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチド又は配列番号２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
    　（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
    　（ｃ）配列番号５の塩基配列で表わされるオリゴヌクレオチド又は配列番号５の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチド又は配列番号６の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
    　（ｄ）配列番号７の塩基配列で表わされるオリゴヌクレオチド又は配列番号７の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチド又は配列番号８の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、及び
    　（ｅ）配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット。     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 oligonucleotide represented by a nucleotide sequence in which one or two bases have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3; An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P. falciparum, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence of SEQ ID NO: 4 lacking one or two bases 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. falciparum, and 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 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 nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 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 an oval malaria parasite, and (e) SEQ ID NO: 9 1 in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 9 Or an oligonucleotide represented by a nucleotide sequence in which 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 Salmonaria parasite, An oligonucleotide represented by a nucleotide sequence 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: 10, and the cox3 gene sequence of the protozoan parasite A primer set comprising an oligonucleotide for obtaining an amplification product of a specific region.
  2. 　工程（１）において、
    　（ｆ）配列番号１１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又は配列番号１２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
    からなるプライマーセットを用いて、プラスモジウム属の核酸を属特異的に増幅し、
    　工程（２）において、工程（１）で増幅された核酸に基づいてプラスモジウム属を検出することをさらに含む請求項１に記載の方法。     In step (1),
    (F) 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 Alternatively, 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 Amplified to
    The method according to claim 1, further comprising detecting Plasmodium based on the nucleic acid amplified in step (1) in step (2).
  3. 　工程（１）は、
    　（ｇ）配列番号１３の塩基配列で表わされるオリゴヌクレオチド又は配列番号１３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１４の塩基配列で表わされるオリゴヌクレオチド又は配列番号１４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いて、前記検体から取得したＤＮＡを鋳型として、１次増幅産物を得る工程と、
    　前記（ａ）～（ｅ）のプライマーセット、又は（ｆ）が存在する場合には（ａ）～（ｆ）のプライマーセットを用いて、前記１次増幅産物を鋳型として、２次増幅産物を得る工程とを含む
    請求項１又は２に記載の方法。     Step (1)
    (G) 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. Obtaining a primary amplification product,
    When the primer set (a) to (e) or (f) is present, the primer set (a) to (f) is used, and the secondary amplification product is obtained using the primary amplification product as a template. The method of Claim 1 or 2 including the process of obtaining.
  4. 　前記１次増幅産物を得る工程及び前記２次増幅産物を得る工程を１回の反応で行う、請求項３に記載の方法。 The method according to claim 3, wherein the step of obtaining the primary amplification product and the step of obtaining the secondary amplification product are carried out in a single reaction.
  5. 　前記工程（１）が、前記（ａ）～（ｅ）であるプライマーセット、（ｆ）が存在する場合には（ａ）～（ｆ）であるプライマーセット、又は（ｇ）が存在する場合には（ａ）～（ｅ）及び（ｇ）であるプライマーセット若しくは（ａ）～（ｇ）であるプライマーセットを一つの反応液中に混合した状態で増幅することを含む請求項１～４のいずれか一項に記載の方法。 In the 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 5. The method according to claim 1, comprising amplifying the primer set (a) to (e) and (g) or the primer set (a) to (g) in a mixed state in one reaction solution. The method according to any one of the above.
  6. 　前記（ａ）～（ｅ）のプライマーセットのそれぞれ、又は（ｆ）が存在する場合には（ａ）～（ｆ）のプライマーセットのそれぞれは、一方のオリゴヌクレオチドにタグ配列が取り付けられ、他方のオリゴヌクレオチドに標識物質又は標識物質導入材料が取り付けられている請求項１～５のいずれか一項に記載の方法。 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, The method according to any one of claims 1 to 5, wherein a labeling substance or a labeling substance introduction material is attached to the oligonucleotide.
  7. 　配列番号１の塩基配列で表わされるオリゴヌクレオチドと配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた熱帯熱マラリア原虫の検出方法。 A method for detecting Plasmodium falciparum using a primer set 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.
  8. 　配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いた三日熱マラリア原虫の検出方法。 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. malaria 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 Alternatively, 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.
  9. 　配列番号５の塩基配列で表わされるオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた四日熱マラリア原虫の検出方法。 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.
  10. 　配列番号７の塩基配列で表わされるオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチドとからなるプライマーセットを用いた卵形マラリア原虫の検出方法。 A method for detecting an oval malaria parasite using a primer set comprising 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.
  11. 　配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセットを用いたサルマラリア原虫の検出方法。 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 have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 9; 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.
  12. 　配列番号１の塩基配列で表わされるオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチドとからなる熱帯熱マラリア原虫検出用プライマーセット。 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.
  13. 　配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなる三日熱マラリア原虫検出用プライマーセット。 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. malaria 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 Alternatively, 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.
  14. 　配列番号５の塩基配列で表わされるオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチドとからなる四日熱マラリア原虫検出用プライマーセット。 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.
  15. 　配列番号７の塩基配列で表わされるオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチドとからなる卵形マラリア原虫検出用プライマーセット。 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.
  16. 　配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるサルマラリア原虫検出用プライマーセット。 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 have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 9; 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 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.
  17. 　一方のオリゴヌクレオチドにタグ配列が取り付けられ、他方のオリゴヌクレオチドに標識物質又は標識物質導入材料が取り付けられている請求項１２～１６のいずれか一項に記載のプライマーセット。 The primer set according to any one of claims 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.
  18. 　下記（ａ）～（ｅ）の５種類のプライマーセット、ＤＮＡポリメラーゼ、及びｄＮＴＰを含む、熱帯熱マラリア原虫、三日熱マラリア原虫、四日熱マラリア原虫、卵形マラリア原虫、及びサルマラリア原虫からなる群より選択される１種以上の検出用キット。
    　（ａ）配列番号１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１の塩基配列において１個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号２の塩基配列で表わされるオリゴヌクレオチド又は配列番号２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ熱帯熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
    　（ｂ）配列番号３の塩基配列で表わされるオリゴヌクレオチド又は配列番号３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号４の塩基配列で表わされるオリゴヌクレオチド又は配列番号４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ三日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
    　（ｃ）配列番号５の塩基配列で表わされるオリゴヌクレオチド又は配列番号５の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号６の塩基配列で表わされるオリゴヌクレオチド又は配列番号６の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ四日熱マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、
    　（ｄ）配列番号７の塩基配列で表わされるオリゴヌクレオチド又は配列番号７の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号８の塩基配列で表わされるオリゴヌクレオチド又は配列番号８の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつ卵形マラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット、及び
    　（ｅ）配列番号９の塩基配列で表わされるオリゴヌクレオチド又は配列番号９の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１０の塩基配列で表わされるオリゴヌクレオチド又は配列番号１０の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつサルマラリア原虫のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドとからなるプライマーセット。     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: 3 or an oligonucleotide represented by a nucleotide sequence in which one or two bases have been deleted, inserted or substituted in the nucleotide sequence of SEQ ID NO: 3; An oligonucleotide for obtaining an amplification product of a specific region of the cox3 gene sequence of P. falciparum, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence of SEQ ID NO: 4 lacking one or two bases 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. falciparum, and 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 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 nucleotide sequence of SEQ ID NO: 8 or the nucleotide sequence of SEQ ID NO: 8 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 an oval malaria parasite, and (e) SEQ ID NO: 9 1 in the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 9 Or an oligonucleotide represented by a nucleotide sequence in which 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 Salmonaria parasite, An oligonucleotide represented by a nucleotide sequence 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: 10, and the cox3 gene sequence of the protozoan parasite A primer set comprising an oligonucleotide for obtaining an amplification product of a specific region.
  19. 　（ｆ）配列番号１１の塩基配列で表わされるオリゴヌクレオチド又は配列番号１１の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１２の塩基配列で表わされるオリゴヌクレオチド又は配列番号１２の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
    からなるプライマーセットをさらに含む請求項１８に記載の検出用キット。     (F) 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 The detection kit according to claim 18, further comprising 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. .
  20. 　（ｇ）配列番号１３の塩基配列で表わされるオリゴヌクレオチド又は配列番号１３の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと、配列番号１４の塩基配列で表わされるオリゴヌクレオチド又は配列番号１４の塩基配列において１個又は２個の塩基が欠失、挿入若しくは置換された塩基配列で表わされるオリゴヌクレオチドであってかつプラスモジウム属のｃｏｘ３遺伝子配列の特定領域の増幅産物を得るためのオリゴヌクレオチドと
    からなるプライマーセットをさらに含む、請求項１８又は１９に記載の検出用キット。     (G) 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 Or 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 a Plasmodium cox3 gene sequence. Detection kit.
  21. 　前記（ａ）～（ｅ）であるプライマーセット、（ｆ）が存在する場合には（ａ）～（ｆ）であるプライマーセット、又は（ｇ）が存在する場合には（ａ）～（ｅ）及び（ｇ）であるプライマーセット若しくは（ａ）～（ｇ）であるプライマーセットを一つの反応液中に混合した状態で、前記（ａ）～（ｅ）又は（ａ）～（ｆ）のプライマーセットを用いた増幅を行う請求項１８～２０のいずれか一項に記載の検出用キット。 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 above (a) to (e) or (a) to (f) The detection kit according to any one of claims 18 to 20, wherein amplification is performed using a primer set.
  22. 　前記（ａ）～（ｅ）のプライマーセットのそれぞれ、又は（ｆ）が存在する場合には（ａ）～（ｆ）のプライマーセットのそれぞれは、一方のオリゴヌクレオチドにタグ配列が取り付けられ、他方のオリゴヌクレオチドに標識物質又は標識物質導入材料が取り付けられている請求項１８～２１のいずれか一項に記載の検出用キット。 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, The detection kit according to any one of claims 18 to 21, wherein a labeling substance or a labeling substance introduction material is attached to the oligonucleotide.
PCT/JP2018/013219 2017-03-31 2018-03-29 Multiplex detection method for 5 types of malarial parasite WO2018181703A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017070144A JP2020096533A (en) 2017-03-31 2017-03-31 Multiplex detection method for five kinds of malarial parasites
JP2017-070144 2017-03-31

Publications (1)

Publication Number Publication Date
WO2018181703A1 true WO2018181703A1 (en) 2018-10-04

Family

ID=63677498

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/013219 WO2018181703A1 (en) 2017-03-31 2018-03-29 Multiplex detection method for 5 types of malarial parasite

Country Status (2)

Country Link
JP (1) JP2020096533A (en)
WO (1) WO2018181703A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110331221A (en) * 2019-08-19 2019-10-15 昆明医科大学 Plasmodium gene diagnostic primers
CN110468224A (en) * 2019-07-02 2019-11-19 贵州省疾病预防控制中心 Plasmodium specifically folds primer PCR molecule diagnosis kit and application method
CN114540526A (en) * 2022-03-23 2022-05-27 福建医科大学孟超肝胆医院(福州市传染病医院) Primer, probe and method for typing detection of five input plasmodium

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013039228A1 (en) * 2011-09-14 2013-03-21 日本碍子株式会社 Method for detecting target nucleic acid
JP2016214101A (en) * 2015-05-15 2016-12-22 国立大学法人東北大学 Simple and quick inspection method of oral bacteria

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013039228A1 (en) * 2011-09-14 2013-03-21 日本碍子株式会社 Method for detecting target nucleic acid
JP2016214101A (en) * 2015-05-15 2016-12-22 国立大学法人東北大学 Simple and quick inspection method of oral bacteria

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ECHEVERRY DF ET AL.: "Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene", MALARIA JOURNAL, vol. 15, no. 128, 29 February 2016 (2016-02-29), XP055612868, ISSN: 1475-2875 *
ISOZUMI R ET AL.: "Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene", PARASITOLOGY INTERNATIONAL, vol. 64, no. 3, 22 September 2014 (2014-09-22) - June 2015 (2015-06-01), pages 304 - 308, XP055612861, ISSN: 1383-5769 *
SAITO T ET AL.: "Rapid and sensitive multiplex single-tube nested PCR for identification of five human Plasmodium species", PARASITOLOGY INTERNATIONAL, vol. 67, no. 3, 31 January 2018 (2018-01-31), pages 277 - 283, XP055612874, ISSN: 1383-5769 *
TIAN L ET AL.: "Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota", BIOMED RES. INT., 2014, pages 180323, ISSN: 2314-6133 *
ZEAITER Z ET AL.: "Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum", J. CLIN. MICROBIOL., vol. 41, no. 3, March 2003 (2003-03-01), pages 919 - 925, XP055612871, ISSN: 0095-1137 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110468224A (en) * 2019-07-02 2019-11-19 贵州省疾病预防控制中心 Plasmodium specifically folds primer PCR molecule diagnosis kit and application method
CN110331221A (en) * 2019-08-19 2019-10-15 昆明医科大学 Plasmodium gene diagnostic primers
CN110331221B (en) * 2019-08-19 2023-05-02 昆明医科大学 Plasmodium gene diagnosis primer
CN114540526A (en) * 2022-03-23 2022-05-27 福建医科大学孟超肝胆医院(福州市传染病医院) Primer, probe and method for typing detection of five input plasmodium
CN114540526B (en) * 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) Primers, probes and method for typing detection of five input plasmodium

Also Published As

Publication number Publication date
JP2020096533A (en) 2020-06-25

Similar Documents

Publication Publication Date Title
Padley et al. Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present
Iseki et al. Evaluation of a loop-mediated isothermal amplification method as a tool for diagnosis of infection by the zoonotic simian malaria parasite Plasmodium knowlesi
WO2018181703A1 (en) Multiplex detection method for 5 types of malarial parasite
US10731208B2 (en) Microfluidic device for detecting target gene, method for manufacturing same, and method for detecting using same
ES2656958T3 (en) Measurement of telomere length in samples fixed in formalin, embedded in paraffin (FFPE) by quantitative PCR
JP5511056B2 (en) Nucleic acid probes and methods for detecting Plasmodium parasites
WO2017212904A1 (en) Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined
Yamamura et al. Evaluation of a new rapid molecular diagnostic system for Plasmodium falciparum combined with DNA filter paper, loop-mediated isothermal amplification, and melting curve analysis
BRPI0721095A2 (en) SYSTEMS AND METHODS FOR MULTIPLEX REAL-TIME PCR ANALYSIS
JP2006201062A (en) Nucleic acid detection or determination method
US7223568B2 (en) Methods for determining nucleotide sequences of single nucleic acid molecules
JP2005172789A (en) Assay system, kit and method for detecting microorganism
Oyhenart et al. Loop mediated isothermal amplification of 5.8 S rDNA for specific detection of Tritrichomonas foetus
JP6844613B2 (en) Primer set for Chlamydia trachomatis detection, Chlamydia trachomatis detection method using this, and reagent kit for that
JP6387606B2 (en) Nucleic acid detection method
JP6623324B2 (en) Multi-item simultaneous detection method for isothermal amplification reaction products
CN110331221B (en) Plasmodium gene diagnosis primer
JP6507791B2 (en) Nucleic acid detection method
AU679725B2 (en) Detection of malaria
JP6977978B2 (en) Method for detecting target nucleic acid
St. Amand et al. Use of specific rRNA oligonucleotide probes for microscopic detection of Mycobacterium avium complex organisms in tissue
Pian et al. Sandwich hybridization-based loop-mediated isothermal amplification (SHB-LAMP) for high-throughput detection of malaria RNA from asymptomatic infections
CN102171369A (en) Method for measuring survivin mRNA
JP6616574B2 (en) Visually identifiable genetic test
Othman et al. Detection of malaria in healthy blood donors using PCR in an endemic area in Yemen

Legal Events

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

Ref document number: 18778279

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18778279

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP