WO1994020613A1 - Depistage du paludisme - Google Patents

Depistage du paludisme Download PDF

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Publication number
WO1994020613A1
WO1994020613A1 PCT/JP1994/000324 JP9400324W WO9420613A1 WO 1994020613 A1 WO1994020613 A1 WO 1994020613A1 JP 9400324 W JP9400324 W JP 9400324W WO 9420613 A1 WO9420613 A1 WO 9420613A1
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Prior art keywords
nucleic acid
malaria
primer
acid fragment
sequence
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PCT/JP1994/000324
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English (en)
Japanese (ja)
Inventor
Yusuke Wataya
Akio Yamane
Original Assignee
Wakunaga Pharmaceutical Co., Ltd.
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Filing date
Publication date
Application filed by Wakunaga Pharmaceutical Co., Ltd. filed Critical Wakunaga Pharmaceutical Co., Ltd.
Priority to US08/513,846 priority Critical patent/US5792609A/en
Priority to AU61159/94A priority patent/AU679725B2/en
Priority to EP94907704A priority patent/EP0694612A4/fr
Publication of WO1994020613A1 publication Critical patent/WO1994020613A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—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
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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 nucleic acid fragment specific to oval malaria parasite and Z or vivax malaria parasite, and P. falciparum malaria parasite, vivax malaria parasite and / or egg using the nucleic acid fragment.
  • the present invention relates to a method for detecting malaria parasites simultaneously or separately.
  • Malari is an infectious disease caused by Plasmodium protozoan infection and is transmitted by Anopheles mosquitoes. Malaria is not only widespread throughout the tropics but also occurs in many temperate regions, with an estimated 270 million cases and an estimated annual deaths from the World Health Organization (WHO). 2 million people (Wo rld Health Organization: Malari a.
  • Serodiagnosis is the second most widely used technique after microscopy. Serodiagnosis is a method that detects antibodies produced after infection with R. parasite (not strong enough to stop the infection, but is said to reduce the symptoms of the infection). I SA (enz yme 1 inkedi mmun osorbent Assays) have been developed. Because these methods are capable of processing large amounts of samples, they are still widely used as a means of mass screening and epidemiological research. However, even if serodiagnosis shows that the antibody is intestinal, there is a problem in that it is not possible to distinguish whether it indicates an infection at the time of the test or an earlier infection [B ruce— ch hwa tt, LJ, Lancet
  • the malaria parasite gene has a characteristic sequence consisting of a large number of repetitions with a length of 20 to 30 bases, and a probe targeting that region is developed, and other regions are used as probes. It has been reported that the sensitivity is dramatically increased [Robert, H. Science 2 31, 144 3 4-1 3 4 6 (1 986)].
  • detection requires a hybridization method that uses a membrane that requires complicated operations, and it has problems such as not being able to obtain the sensitivity as expected when actually tested in a clinical setting (L Amar, DE Am. J. Trop. Med. Hy g. 34, 663-667 (1 985)].
  • the present inventors have proposed a nucleic acid fragment specific for Plasmodium falciparum, a nucleic acid fragment specific for Plasmodium vivax, a nucleic acid fragment specific to both of them, and a Plasmodium falciparum using the same.
  • a method for detecting Z or vivax malarial parasite and filed a patent application earlier (Japanese Patent Application No. 4-365485).
  • oval malarial parasites and vivax malaria parasites are known.
  • an object of the present invention is to provide a method for simultaneously detecting an oval or Plasmodium vivax malaria parasite-specific nucleic acid fragment capable of detecting only an oval or Plasmodium vivax, and simultaneously using four malaria parasites using the same. It is an object of the present invention to provide a method of detecting the difference.
  • the present invention relates to a nucleotide sequence represented by the following formula (1) (SEQ ID NO: 1), a nucleic acid fragment containing a nucleotide sequence complementary thereto or a mutant sequence thereof, and an oval malarial parasite comprising the nucleic acid fragment: It provides a primer or a probe.
  • the present invention provides a nucleic acid fragment comprising a base sequence represented by the following formula (2) (SEQ ID NO: 2), a complementary base sequence or a mutant sequence thereof, and an oval malaria parasite comprising the nucleic acid fragment: It provides a detection primer or probe.
  • the present invention provides a nucleic acid fragment comprising a base sequence represented by the following formula (3) (SEQ ID NO: 3), a complementary base sequence or a mutant sequence thereof, and It is intended to provide a primer or probe for detecting Malaria parasites.
  • the present invention provides a base sequence represented by the following formulas (1), (3), (4), (5), and (6) (SEQ ID NOs: 1, 3, 4, 5, and 6, respectively), which is complementary thereto.
  • the present invention provides a method for identifying P. falciparum malaria, M. vivax, F. malaria and oval malaria using a nucleic acid fragment containing a basic nucleotide sequence or a mutant sequence thereof as a probe D
  • the present invention provides a primer for detecting malaria parasite comprising a nucleic acid fragment containing a base sequence represented by the following formulas (7) to (10) (respectively, SEQ ID NOs: 7 to 10) or a mutant sequence thereof. Is what you do.
  • nucleic acid fragments having the above nucleotide sequence of the present invention are used alone or in combination as a primer or as a probe, all of the malaria parasites that infect humans, that is, P. falciparum, M. vivax, Plasmodium falciparum and oval malaria parasites can be detected simultaneously or simply, quickly, with high sensitivity, or accurately identified. Therefore, it can be used very advantageously for malarial treatment or large-scale mass screening in endemic areas.
  • FIG. 1 shows Plasmodium falciparum 18S ribosomal RNA gene, Plasmodium vivax 18S liposome RNA gene, Plasmodium vivax 18S S liposomal RNA gene, Oval malaria parasite 18
  • FIG. 2 is a view showing a comparison of partial sequences of an S ribosomal RNA gene and a human 18 S ribosomal RNA gene. In the figure, one indicates a deletion. Numbers in parentheses indicate base sequence numbers. Underlines indicate regions corresponding to the respective protozoan-specific primers or probes [Equations (1), (3), (4), (5)]. 1, 4, 2, and 5 are regions corresponding to the primers represented by formulas (7), (8), (9), and (10). 3 is the area corresponding to the common probe shown in equation (6).
  • nucleic acid fragments represented by the formulas (1), (3) to (10) in the present invention can be classified as follows according to their properties.
  • (C) A nucleic acid fragment that selectively detects or amplifies only the 18S ribosome RNA gene of P. falciparum.
  • (E) a nucleic acid fragment that detects or amplifies any of the 18S ribosome RNA genes of Plasmodium falciparum, Plasmodium vivax, Plasmodium vivax, and Oval malaria parasite.
  • nucleic acid fragments can be synthesized by an automatic DNA synthesizer.
  • the nucleotide sequence of the nucleic acid fragment of the formula (1) was determined as follows. First, blood was collected from a patient infected with oval malaria parasite, and the resulting DNA was subjected to a gene amplification reaction using the primer of formula (7) and the primer of formula (8). An amplified product of 40 bp was obtained, and the nucleotide sequence of the amplified product was determined. As a result, the amplified product was found to have the following formula (2).
  • the nucleic acid fragment having the nucleotide sequence of the formula (1) is not present in the Plasmodium falciparum, Malaria vivax, and Malaria vivax malaria parasites, and is not present in the oval malaria parasite.
  • the S ribosomal RNA gene is selectively detected or amplified.
  • nucleic acid fragment of the formula (3) was synthesized by a DNA automatic synthesizer, and the 18S ribosome RNA gene of Plasmodium vivax was selectively detected or amplified by a gene amplification reaction. Similarly, the reactivity of the nucleic acid fragments of formulas (4) to (6) was confirmed.
  • the mutant sequence in the nucleic acid fragment of the present invention may be any one that functions as a primer or probe in the detection of P. falciparum, P. vivax, R. vivax and / or oval malaria parasite. Any of them may be used. For example, it may be one in which some bases in the basic base sequence represented by the above formulas (1) to (10) are deleted or substituted or added with other bases. More specifically, examples thereof include a base sequence corresponding to a substantial portion of the same gene in the mutant strain of malaria parasite, an RNA sequence corresponding to these genes, and the like. However, in the vicinity of the 3 'end of the primer, which is expected to greatly affect the efficiency of the primer extension reaction, it is preferable that no mutation is present or that the mutation is minimized, and more preferably, it is 5 'Make the mutation near the end.
  • nucleic acid fragment of the present invention In order to detect the four types of malaria parasites using the nucleic acid fragment of the present invention as a primer or a probe, it is preferable to use these nucleic acid fragments as a label or a solid-phase carrier-bound derivative.
  • Examples of the label of the nucleic acid fragment of the present invention include those in which a detectable label is bound to the nucleic acid fragment.
  • the labeling substance either a non-radioactive substance or a radioactive substance may be used, but a non-radioactive substance is preferable.
  • Non-radioactive substances include those which can be measured directly, such as fluorescent substances (for example, fluorescein and its derivatives (fluorescein isothiosinate), rhodamine and its derivatives (tetramethylrhodamine isotiosinate, Texas red). Etc.]], chemiluminescent substances (for example, acridine) and substances that emit delayed fluorescence (DTTA: manufactured by Pharmacia).
  • the labeling substance may be, for example, biotin or hapten.
  • biotin avidin or streptavidin specifically binds thereto, and in the case of hapten, an antibody which specifically binds to avidin or streptavidin.
  • the hapten digoxigenin, a compound having a 2,4-dinitrofujyl group, can be used, and further, biotin or a fluorescent substance can be used as the hapten. Any of these signs may be used alone or, if necessary, in a combination of a plurality of kinds by known means (see JP-A-59-93098, JP-A-59-93099). ) Can be introduced into the primer.
  • Examples of the solid phase carrier-binding derivative of the nucleic acid fragment of the present invention include those in which a site capable of binding to the solid phase carrier is introduced into the nucleic acid fragment of the present invention.
  • a site capable of binding to the solid phase carrier solid phase carrier binding site
  • the above-mentioned non-radioactive labeling substance can also be used.
  • Preferable specific examples thereof include a fluorescent substance such as piotin or fluorescein, a compound having a 2,2-dinitrophenyl group or a hapten such as digoxigenin, and these may be used alone or in combination of plural kinds if necessary. It can be introduced into the nucleic acid fragment of the present invention in advance.
  • the site and the label for detection are not the same.
  • the solid phase carrier referred to here is inert to the solvent used for the reaction and all the reagents, can be separated from the reagent solution by any method, and can be selectively bonded to the above site. is there.
  • examples of such a substance include those obtained by introducing a streptovidine or an antibody capable of capturing the above-mentioned site into a solid phase material such as microtiter beads, polystyrene balls, polygalrose beads, and polyacryl beads.
  • it is a microtiter tube excellent in operability, mechanization and the like.
  • a carrier having streptavidin bound to a solid phase a fluorescein residue or a 2,4-dinitrophenyl group may be used.
  • a carrier in which an antibody for each is bound to a solid phase can be used.
  • dot-plot hybridization, Southern hybridization, etc. can be used.
  • RNA can be used as the target substance for detection.
  • sensitivity is increased by targeting 18S liposome RNA, which has a high copy number per cell. be able to.
  • various gene amplification reactions can be used, and sensitivity can be dramatically increased by targeting these amplified products.
  • the PCR method can be used as a gene amplification reaction to perform dot-blot hybridization and reverse-dot hybridization targeting the amplified product [ITT nis, MA, PCR. Ac ad emi c Press (1 990)].
  • a method using a microtiter well as disclosed in Japanese Patent Application No. 182035/1991 is more practical.
  • Detection of four types of malaria parasites by a gene amplification reaction using the nucleic acid fragment of the present invention as a primer is basically performed as follows.
  • the primer selected from the above formulas (7) to (10) or the formula (2) After a gene amplification reaction using a primer selected from the nucleotide sequences shown, the presence or absence of a gene amplification product of a desired size may be examined by agarose gel electrophoresis or the like. More simply, as disclosed in Japanese Patent Application No.
  • a gene amplification reaction is carried out using a primer in which a labeled substance is introduced into one primer and a solid phase carrier binding site is introduced into the other primer, and ELISA ( EnzymeLinkedImmunoSorbent Assay) can be detected.
  • ELISA EnzymeLinkedImmunoSorbent Assay
  • the gene amplification product using the primers of the above formulas (1), (3) to (6) or the primers selected from the base sequence represented by the formula (2) is used. Due to subtle differences in the chain length between malaria species, or by subjecting the gene amplification reaction product to restriction enzyme treatment, You can take advantage of what happens. More simply, a gene amplification reaction is carried out with a primer selected from the formulas (7) to (10) or a primer selected from the base sequence represented by the formula (2), and the amplified product is treated as a target with the formula This is a method of detecting with a probe selected from (1), (3) to (6) or a probe selected from the base sequence represented by formula (2).
  • a gene amplification reaction is carried out using a primer selected from the formulas (6) to (10) or a primer selected from the base sequence represented by the formula (2), into which a labeling substance has been introduced.
  • This is a method in which hybridization is performed with each of the probes (1), (3) to (5) fixed to a microtiter well to detect.
  • Primary samples include blood obtained from infected individuals or patients.
  • the collected blood may be used after removing serum components or as it is (e.g., using a surfactant such as savonin [Biooch em. Biophys.Res.shiomom.175, 179-184 ( 1)
  • the blood cell components can be destroyed using)] and the blood cell components can be removed by centrifugation.
  • Protozoa from which blood cells have been removed can be destroyed by treatment with proteinase or the like in the same manner as ordinary cells.
  • the primer of the present invention By adding the primer of the present invention to the above sample, the target mala If L. parasites are present, a gene amplification reaction based on the primer extension reaction can be performed.
  • the extension reaction of the primer is carried out by four types of nucleotide triphosphates ((doxy-denosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate (a mixture of these is called dNTP). )] As a substrate and incorporated into the primer.
  • E.c01i DNA polymerase I Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase and the like are used.
  • a thermostable enzyme such as Taq polymerase capable of performing an elongation reaction at a high temperature can enhance the specificity of target sequence recognition by a primer (for details, see JP-A-11-314965 and JP-A-11-252300). See).
  • Two types of primers according to the present invention [for example, a combination of (7) and (8), a combination of (9) and (10), a combination of (7) and (10) or a combination of (9) and (8)
  • the target malaria parasite 18 S liposomal RNA gene can be efficiently amplified.
  • Detection of a conjugate between the target gene and the detection primer generated by the primer extension reaction enables detection of the malaria parasite in the sample, but a preferred method for detecting the conjugate is the primer extension reaction described above. It depends on the type or form of the product, that is, its synthetic nucleic acid.
  • the gene amplification reaction product is double-stranded, and this synthetic nucleic acid is electrophoresed [Saiki, RK Science 230, 135-350-(1985)] or a labeled probe.
  • the method can be detected by the dot hybridization method CSaiki, RK Nature 324, 163-166 (1986)].
  • a reverse dot hybridization method for hybridizing the probe immobilized on the solid phase carrier and the labeled synthetic nucleic acid chain [Saiki, RKProc. Natl. USA, 86, 6 230-6 234 (1 89 9)]
  • a column method using a solid adsorbent see Japanese Patent Application Laid-Open No. 11-34965).
  • nucleic acids can be detected more easily. That is, there is a method of using the combination of the solid phase carrier-binding derivative and the label as a primer, bringing the reaction product obtained by the gene amplification reaction into contact with the solid phase carrier, and then washing and removing impurities with an appropriate solvent. Also in this method, the synthetic nucleic acid strand extended from the primer having the solid-phase carrier binding site forms a double strand with the synthetic nucleic acid extended from the other primer-introduced primer, and The product of the amplification reaction is immobilized on the solid-phase carrier in a form having a label, and is specifically immobilized.
  • JP-A-5-227998 it is very simple and practical to use the method disclosed in JP-A-5-227998. That is, a gene amplification reaction was carried out using a primer into which the above-mentioned non-radioactive label was introduced, and the reaction product was used in a microphone ⁇ titer obtained by immobilizing the nucleic acid fragments represented by formulas (6) to (10) in advance. This is a method for hybridization in the ell. Only when a sequence complementary to the solid-phased nucleic acid fragment is present in the microtiter well, the gene amplified product will bind to the microtiter well, and the labeled product bound to the primer may be detected.
  • a general method may be used for detection of the labeling substance according to the labeling substance to be used.
  • the labeling substance is a radioisotope
  • the activity may be measured as it is.
  • the substance is biotin, avidin (or streptavidin) monoenzyme conjugate, if it is a hapten
  • the antibody monoenzyme conjugate Can be reacted with a substrate to obtain a component that can be detected by color or fluorescent means.
  • the labeling substance is fluorescent
  • the intensity may be measured using a fluorometer as it is.
  • the deoxyoligonucleotide was prepared by the phosphoramidite method of Carruthers et al. : 0.2 micromolar scale using Applied Biosystems DNA synthesizer model 381 A based on Te trahedron Lett., 22, 1859 (19981) 3 I went in.
  • those having a labeled substance or solid-phase carrier binding site introduced are first synthesized as oligonucleotides having an amino group introduced at the 5 'end of each, and then the labeled substance or solid-phase carrier binding is performed using an appropriate reagent. The site was introduced. An example is shown below.
  • an oligonucleotide (5 'GAACGAAAGTTA AGGGAGT) having an amino group introduced at the 5' end was synthesized using an automated DNA synthesizer model 381A of Applied Biosystems.
  • Protected mononucleotide phosphoramidite was sequentially added to 0.2 ⁇ mol of the support, and the base at the 5 ′ end was added, followed by Aminolink II (trade name: Applied Biosystems).
  • Aminolink II trade name: Applied Biosystems
  • Aqueous completion amination oligonucleotide de was roughly purified by gel filtration (1.0 ⁇ D.:. 1 8 0 / ⁇ ⁇ ) to 1 MN a HC_ ⁇ 3 (0 / ⁇ ) was added, to which 5% ( v / v) A solution of dinitrofluorobenzene in ethanol (100 £) was added, and the mixture was heated at 37 for 2 hours. After the reaction, the reagents were removed by gel filtration in the same manner as for the biotinylated oligonucleotide, and purified by reverse-phase HPLC (yield: 0.38 : .D.).
  • Ovarian malaria parasite genes were extracted from the blood of patients determined to be infected with oval malaria parasites by observation of blood smears by an operation of £ TF.
  • blood 5 ml
  • oval malaria parasite was suspended in PBS and centrifuged at 300 Orpm for 10 minutes.
  • the supernatant was removed, and the blood cell component was suspended in 0.15% (w / v) saponin in PBS solution (7.5 rd) and allowed to stand at 37 t for 20 minutes.
  • PBS was added to bring the volume to 45 mL, and the mixture was centrifuged at 300 O rpm for 15 minutes.
  • the precipitate was suspended in PBS (45 ml) and centrifuged similarly. This operation was repeated twice more.
  • the precipitate was suspended in PBS (50 ⁇ ) and transferred to a 2 ml centrifuge tube. This was centrifuged at 10,000 rpm for 5 minutes to remove the supernatant, and the precipitate was washed with 10 mM Tris-HC pH 7.5, 1 mM EDTA pH 8.0, 0.1 M Na C £ (1.r ) And further added 20% SDS (37.5 ⁇ ) andconductivease K (10 jug: 30 ⁇ ), and mixed by inversion at room temperature for 1 hour. Further, it was left still at 37 for 16 hours. To this, 1Z3 volume of saturated saline was added, mixed by inversion, allowed to stand at room temperature for 15 minutes, and then centrifuged at 1500 rpm for 15 minutes.
  • the double-stranded fragments were ligated using T4 DNA ligase, and then blunt-ended using a Klenow fragment of E. coli DNA polymerase I and four types of deoxyribonucleotide triphosphates.
  • plasmid pUC-SfiX2 Japanese Unexamined Patent Publication No. 2-190194 was cut with a restriction enzyme BamHI and blunt-ended with a K1enow fragment.
  • These DNAs were ligated using T4 DNA ligase to transform Escherichia coli DH5.
  • the desired clone was selected by restriction enzyme analysis.
  • the obtained plasmid was cleaved with a restriction enzyme SfiI, and a portion containing the repeat of the sequence was purified and recovered by electrophoresis.
  • an S fi fragment containing an excessive amount of the sequence repetition with respect to the vector was ligated with T4 DNA ligase.
  • pUC-SfiX2 cut with SfiI was used as a vector.
  • MV118 was transformed, and clones containing about 60 unit sequences shown in the above (A), (B) and (C) were selected by restriction enzyme analysis.
  • the obtained single-stranded DNA was adjusted to a concentration of 1 igZ50 ⁇ £ with 10 mM Tris-HCPH7.6 and 1 mM EDTA solution, and an equal volume of immobilized buffer (1.5 M NaC i. 0.3 MT ris -HC ⁇ (PH 8.0), 0.3 M MgC £ 2 ), mix and add to microtiter pellets (Sumitomo Beta Light, Sepaplate 8 FH type) 1 0 0 // ⁇ ⁇ was added. ⁇ The lid was covered and left for 37 t for 16 hours.
  • a microtiter well for detecting protozoa and a microtiter well for immobilizing single-stranded DNA containing the unit sequence of (C) were used to detect all four protozoa.
  • the microphone ⁇ titer was placed in a vinyl pack, sealed and stored at 4 t.
  • Plasmodium falciparum (0.22 ng / x, 5 ⁇ ⁇ )
  • B Plasmodium vivax (DNA corresponding to 1 0 4 parasites isolated from patients)
  • C DNA solution obtained in oval malaria Ryo protozoa (example 2, 5
  • D normal human DNA obtained from leukocytes (100 ng / fi i, 5 ⁇ ⁇ )
  • Each DNA solution was added to 1 ysis solution (11 OmM Tris-HCi (PH8.9), 1.5 mM MgC £ 2 , 8 Om KC £, 500 xg m £ BSA, 0.1% (w / v) sodium cholate, 0.1% Triton X-100 0 200 / gm £ ⁇ tinase K, 0.
  • the plate was washed with about 30 of the buffer described above. This washing operation was performed twice more, 100 ⁇ l of a solution of ⁇ -nitrophenyl phosphate (4 mg / m £, dissolved in 1 M jetanolamine buffer) was added, and the mixture was left at 25 t for 30 minutes.
  • the absorbance at 405 runs was measured with a microplate reader. As a result, as shown in Table 1, all the malaria parasites could be detected by using the primer of the present invention.
  • 1% (w / v) cholate sodium, 500 i / n BSA, 1 UT th Polymerase] was used as a lO x calo and gene amplification reaction (94: 30 seconds, 50: 60 seconds, 72, 60 seconds, 3 0 cycle condition).
  • the mixed solution after the reaction was heat-denatured on a boiling water bath for 5 minutes and quenched on ice.
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

Méthode de dépistage de la fièvre tropicale, du paludisme tierce, du paludisme quarte et des autres accès palustres à Plasmodium ovale par utilisation d'amorces représentées par les formules (1) et (3) à (10) ci-après. Cette méthode permet de dépister immédiatement et rapidement, avec une sensibilité élevée et une différenciation précise, tous les types de plasmodies atteignant l'être humain, permettant ainsi le traitement ou le dépistage à grande échelle du paludisme dans les zones où cette maladie frappe.
PCT/JP1994/000324 1993-03-12 1994-02-28 Depistage du paludisme WO1994020613A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/513,846 US5792609A (en) 1993-03-12 1994-02-28 Detection of malaria
AU61159/94A AU679725B2 (en) 1993-03-12 1994-02-28 Detection of malaria
EP94907704A EP0694612A4 (fr) 1993-03-12 1994-02-28 Depistage du paludisme

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP5052541A JPH06261758A (ja) 1993-03-12 1993-03-12 マラリアの検出
JP5/52541 1993-03-12

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WO1994020613A1 true WO1994020613A1 (fr) 1994-09-15

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US (1) US5792609A (fr)
EP (1) EP0694612A4 (fr)
JP (1) JPH06261758A (fr)
AU (1) AU679725B2 (fr)
WO (1) WO1994020613A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100451398B1 (ko) * 2001-10-09 2004-10-06 (주)바이오니아 열대열말라리아와 삼일열말라리아의 동시 검출방법

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WO1998035057A1 (fr) * 1997-02-06 1998-08-13 The National University Of Singapore Diagnostic de l'infection a plasmodium par analyse de materiel genetique extrachromosomique
KR20020028385A (ko) * 2000-10-09 2002-04-17 박제철 마커를 이용한 말라리아 원충에 감염된 모기 검출방법
ES2471092T3 (es) * 2006-11-30 2014-06-25 Id-Fish Technology, Inc. Sondas de ácido nucleico y métodos para detectar parásitos de Plasmodium
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AU679725B2 (en) 1997-07-10
AU6115994A (en) 1994-09-26

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