JP2003250564A - Plasmodium malaria laveran and its diagnosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain new two kinds of Plasmodium malaria Laveran together with their SSU rRNA gene sequences and to provide a material for malaria diagnosis for these new protozoans. <P>SOLUTION: The prevent invention provides the Plasmodium malaria Laveran in which base sequences of SSU rRNA genes are each a specific sequence, a material for diagnosing malaria infection by these protozoans and a method for diagnosing the malaria. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The invention of the present application relates to a novel Plasmodium vivax and its diagnosis.

[0002]

Malaria is caused by infection with one or more malaria parasites. P. falciparum, P. vivax, P. malariae, and egg malaria (P. ovale) are known as malaria parasites. These protozoa invade and infect the human body in the form of sporozoite from their salivary glands by biting of female mosquitoes of the genus Anopheles.

Malaria is a multiple infectious disease in the world next to acute lower respiratory tract infections, AIDS and diarrhea.
(World Health Organization) estimates 1999
About 45 million cases are affected each year, of which about 1.1 million die (The World Health Report 2000, p.164 and p.17).
0, WHO 2000).

Early detection of malaria measures,
Early treatment is of the utmost importance, for which mass diagnosis is being carried out.

[0005] For the diagnosis of malaria, a microscope method in which a blood smear is stained with Giemsa for examination and a serodiagnosis method for detecting an antibody produced after infection with malaria parasite are widely adopted. However, these frequently used methods have problems that the inspection procedure is complicated and the determination result is not always accurate.

On the other hand, a DNA for detecting a malaria parasite-specific gene sequence by using an oligonucleotide probe by applying a molecular biology technique or PCR amplifying a specific malaria parasite SSU rRNA gene. A diagnostic method has been proposed (for example, Japanese Patent Laid-Open No. 05-227998), and simple and highly accurate malaria diagnosis is expected.

[0007]

As described above, various diagnostic methods have been developed as countermeasures against malaria, and in particular, DNA diagnosis using the probe method or PCR method is effective for malaria diagnosis because of its convenience and accuracy of judgment. It is expected to be the mainstream.

However, even in such a diagnosis by the DNA method, there are problems to be solved in order to further improve the determination accuracy. That is, as described above, the four types of malaria parasites have different types and have different gene sequences. Moreover, the gene sequences currently used for DNA diagnosis do not correspond to all malaria parasites. DNA diagnosis is completely ineffective against an unknown malaria parasite having a gene sequence different from the known gene sequence.

The invention of this application has been made in view of the above circumstances, and it is an object of the invention to provide a novel type of Plasmodium vivax, which has never been known, together with its gene sequence. I am trying.

Another object of the invention of this application is to provide a method for diagnosing this new type Plasmodium vivax and a genetic material therefor.

[0011]

Means for Solving the Problems As an invention for solving the above-mentioned problems, this application discloses two types of new Plasmodium vivax, that is, S. vivax whose nucleotide sequence of SSU rRNA gene is SEQ ID NO: 1. A malaria parasite and a Plasmodium vivax whose nucleotide sequence of SSU rRNA gene is SEQ ID NO: 2 are provided.

The invention of this application also provides a purified polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and a purified polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2.

The invention further provides an oligonucleotide which hybridizes to each of the above-mentioned purified polynucleotides. It is a preferred embodiment that the oligonucleotide is bound with a labeling substance.

The present invention further provides a microarray (DNA chip) provided with the above-mentioned purified polynucleotide or oligonucleotide.

Furthermore, the invention of this application provides antibodies against the above-mentioned two types of Plasmodium vivax.

The present invention further provides a method for diagnosing malaria, which comprises detecting the presence of the Plasmodium vivax in a test sample.

In the present invention, the "SSU rRNA gene" is a gene of a small subunit rRNA (SSU rRNA or 18S rRNA) which is one of the ribosomal components of Plasmodium vivax. In addition, "polynucleotide" and "oligonucleotide" are long and single-stranded nucleotide chains (DNA chain or RNA chain), respectively, and the standard is that the polynucleotide is 100 bp or more and the oligonucleotide is less than 100 bp. However, there are exceptions.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The novel Plasmodium vivax parasites of the present invention (hereinafter sometimes referred to as "new strain 1 strain" and "new strain 2 strain") have SSU rRNA of SEQ ID NO: 1 as described above. And has the base sequence of SEQ ID NO: 2. The nucleotide sequences of the SSU rRNA genes of the new strain 1 and the new strain 2 are the known Plasmodium vivax Uganda-1 strain (Mol. Bio
chem.Parasitol. 45: 281-299, 1991; GenBank Accessio
n No. M54897), Greek strain, LSTMH strain and PNG strain
Several bases differ from each SSU rRNA gene sequence of the (Papua New Guinea) strain (Fig. 1).

These new type 1 and new type 2 strains of Plasmodium vivax are isolated from candidate cells by known means from blood of malaria-infected patients and the like, and known phenol: chloroform extraction from these cells is performed. The chromosomal DNA is isolated by the method and the nucleotide sequence of the SSU rRNA gene region of this chromosomal DNA is determined, and the sequence is the sequence of the new strain 1 (SEQ ID NO: 1) or the sequence of the new strain 2 (SEQ ID NO: 2). It can be obtained by identifying the candidate bacterial cell as the Plasmodium vivax new strain 1 strain or new strain 2 of the present invention. Whether the SSU rRNA gene sequence of the candidate bacterial cell is the same as SEQ ID NO: 1 or SEQ ID NO: 2 is provided by the hybridization method using the oligonucleotide probe provided by the present invention, or by the present invention. It can also be confirmed by a PCR (Polymerase Chain Reaction) method using an oligonucleotide primer set.

The purified polynucleotide of the present invention is a polynucleotide (DNA chain and RNA chain) isolated and purified from the SSU rRNA gene sequence of each of the above-mentioned strains of Plasmodium vivax new strain 1 and new strain 2. The single-stranded form and its complementary strand, and the double-stranded form in which they are paired are included. Such a purified polynucleotide can be obtained, for example, by a known method such as PCR using the chromosomal DNA extracted from the new strain 1 or new strain 2 described above as a template and using the oligonucleotide primer set provided by the present invention. can do. The obtained purified polynucleotide may be subjected to, for example, PCR, NASBN (Nucleic acid seq).
uence based amplification) method, TMA (Transcription-
mediated amplification) method and SDA (Strand Displa)
It can be used after being amplified by a commonly used gene amplification method such as cement amplification method.

The oligonucleotide of the present invention is specifically an oligonucleotide probe used when screening the above-mentioned purified polynucleotide, or an oligonucleotide primer set used when PCR-amplifying the above-mentioned purified polynucleotide. Etc.

The oligonucleotide primer set of the present invention is a set of at least two oligonucleotides for PCR amplification of the above-mentioned purified polynucleotide. This primer set PCR-amplifies the SSU rRNA genes of the new strain 1 and the new strain 2 separately, respectively, and the different sequence regions of the SSU rRNA gene sequences of the new strain 1 and the new strain 2 and other malaria parasites, respectively. It is preferable that the primer sets can be separately PCR-amplified. In addition, other Plasmodium vivax (Uganda-1 strain, Greek strain, LSTMH strain and Papua New Guin strain)
It is particularly preferable that the primer set is capable of separately PCR-amplifying different sequence regions of the respective SSU rRNA gene sequences of the (ea strain). Such a sequence region is
For example, it can be appropriately selected with reference to the base substitution shown in FIG.

The primer set comprises SEQ ID NOS: 1 and 2.
And other malaria parasite SSU rRNA gene sequences can be designed based on the respective nucleotide sequences, and can be prepared through each step of synthesis and purification. Note that, for example, the following points can be pointed out as points to be noted when designing a primer. The size of the primer (the number of bases) is 15-40 bases, preferably 15-30 bases, in consideration of satisfying specific annealing with the template DNA. However, LA (long accurate)
When performing PCR, at least 30 bases are effective. Avoid the complementary sequence between both primers so that the pair of (2) primers consisting of the sense strand (5 'end side) and the antisense strand (3' end side) do not anneal to each other, and have a hairpin structure in the primer Also avoid self-complementary sequences to prevent the formation of Furthermore, mold DN
To ensure a stable bond with A, the GC content should be about 50%,
Make sure that GC-rich or AT-rich is not unevenly distributed in the primer. The annealing temperature is Tm (melting temp
However, in order to obtain highly specific PCR products, primers with Tm values close to each other at 55-65 ° C are selected. It is also necessary to pay attention to adjusting the final concentration of the primer used in PCR to be about 0.1 to about 1 μM. In addition, commercially available software for primer design, such as OligoTM [National Bioscience Inc.
(Made in the United States), GENETYX [made by Software Development Co., Ltd. (Japan)] and the like can also be used.

The oligonucleotide probe is a DNA fragment or RNA fragment that hybridizes to the above-mentioned purified polynucleotide under stringent conditions. For example, it is a continuous 10 to 99 base DNA fragment complementary to a partial region of the base sequence of SEQ ID NO: 1 or 2. Here, the stringent conditions are conditions that allow specific hybridization with the gene or polynucleotide probe described above, salt concentration in the hybridization and washing steps, the concentration of organic solvent (formamide, etc.),
Specified by temperature conditions. For details, see U.S. Patent No.
It is specified in detail in 6,100,037 etc. And this oligonucleotide probe is
While hybridizing to different sequence regions of the SSU rRNA gene, respectively, it is preferable to hybridize individually to the different sequence regions of the SSU rRNA gene sequences of the new strain 1 and the new strain 2 and other malaria parasite strains. In addition, other Plasmodium vivax (Uganda-1 strain, Greek strain, L
SMH r of STMH and Papua New Guinea)
It is particularly preferable that it specifically hybridizes to a sequence region having a different NA gene sequence. The known Plasmodium vivax Uganda-1 strain and Papua New Guinea
A part of each sequence of the strain, Plasmodium falciparum, Plasmodium vivax and Plasmodium falciparum is as shown in FIG. 2, and the probe consisting of the base sequence shown in lowercase letters in FIG. It is known. Therefore, the probe of the present invention can be designed by appropriately selecting from a sequence region different from the known probe shown in FIG.

Further, the oligonucleotide (probe or primer) of the present invention can be bound with a labeling substance by a known method. By binding such a labeling substance to a probe or a primer, it is possible to easily determine the presence or absence of a polynucleotide hybridized with the probe, or the presence or absence of a polynucleotide PCR-amplified by the primer, by detecting a labeling signal. Become. Although non-radioactive substances and radioactive substances can be used as the labeling substance,
Labeling with a non-radioactive substance is preferred. As non-radioactive substances, fluorescent substances [fluorescein and its derivatives (fluorescein isothiocyanate, etc.), rhodamine and its derivatives (tetramethylrhodamine isothiocyanate, Texas red, etc.)], chemiluminescent substances (acridine, etc.) can be directly measured. ), A delayed fluorescent substance (DTTA, etc.) and the like can be used. The labeling signal can also be indirectly detected via a substance that specifically binds to the labeling substance. Examples of the labeling substance in such a case include biotin and hapten, and in the case of biotin, avidin or streptavidin that specifically binds to this, but in the case of hapten, anti-hapten antibody or the like. Available. 2,4-as a hapten
A compound having a dinitrophenyl group or the like can be used, and further, biotin, a fluorescent substance or the like can be used as the hapten. In the case of a primer,
You may make it couple | bond with the polynucleotide which codes green fluorescent protein (GFP) etc.

Microarray (DNA chip) of the present invention
May be a product obtained by directly synthesizing the above-mentioned purified polynucleotide or oligonucleotide on a substrate, or may be an oligonucleotide spotted on a substrate coated with a material to which nucleotides bind. In addition, this microarray preferably includes probes derived from other malaria parasites.

The antibody of the present invention is a polyclonal antibody or a monoclonal antibody that recognizes Plasmodium vivax new strain 1 or new strain 2, and the whole molecule specifically recognizing each strain, Fab, F (ab ') ₂ , Fv fragments, etc. are all included. Such an antibody can be obtained from serum after immunizing an animal with the strain as an antigen. Alternatively, it can be prepared by introducing the above-mentioned purified polynucleotide into the muscle or skin of an animal by injection or gene gun, and then collecting serum. As animals, mice, rats, rabbits, goats, chickens and the like are used. Monoclonal antibodies can be produced by fusing B cells collected from the spleen of an immunized animal with myeloma to prepare a hybridoma.

The method for diagnosing malaria of the present invention is characterized by detecting the presence of a new strain of Plasmodium vivax and a new strain of Plasmodium vivax in a test sample. The test sample is a biological sample such as blood obtained from a malaria infected person or patient, or a resident in a high-risk area of malaria infection.

The detection of the target malaria parasite in the test sample is
For example, serum components from blood, blood cell components are removed to isolate protozoan cells, chromosomal DNA is extracted from the cells by known means, and the DNA is used as a target by the hybridization method using the above-mentioned probe. It can be carried out. Specific examples of the hybridization method using a labeled DNA probe include, for example, Allele-specific Olig.
oligonucleotide probe method, Oligonucleotide Ligation Assa
Known methods such as the y method and the Invader method can be adopted.

Further, a diagnosis is carried out by carrying out PCR amplification using the chromosomal DNA as a template and the above-mentioned primer set, and comparing the nucleotide sequence of the PCR product with SEQ ID NO: 1 or 2 or a partial sequence thereof. be able to. Furthermore, such determination can be performed by, for example, PCR-SSCP method, PCR-
The CFLP method, PCR-PHFA method and the like may be performed. Also, Rolling
Circle Amplification Method, Primer Oligo Base Extensio
A known method of the n method can also be adopted.

Furthermore, the chromosome isolated from the test sample
DNA or a fragment thereof is labeled, and this is applied to the microarray, and the type of the test sample DNA is determined by using the presence or absence of hybridization between the labeled test sample DNA and the oligonucleotide on the substrate as an index. May be.

The diagnostic method of the present invention can also be carried out using the above-mentioned antibody by a known method such as an indirect fluorescent antibody method or an ELIZA method.

The details of the research for identifying the novel strain 1 and the novel strain 2 of the present invention will be described in detail below. Also,
The PCR method and the like carried out in the following studies show one example of the oligonucleotide primer of the present invention, but the invention of this application is not limited to the following examples. 1: Materials and Methods 1-1: Study Area Malaria field survey in Myanmar was carried out during the period from September 1998 to February 2000, Dawei (Tanintharyi Province,
South Myanmar), Myithyina (Kachin Province, Northern), Tachi
lek and Muse (Shan Province, Northeast), Thaton and Kyaik
to (Mon State, South), Pyin Oo Lwin (Mandalay Division, Central), and Than Dwe (Rakhine, Western). Informed consent was obtained from all patients. 1-2: On-site diagnosis with acridine orange (AO) staining A thin finger and a thick smear were prepared by a single finger puncture,
AO method (Kawamoto, 1991, Lancet 337: 200-202; Kawamot
o and Billingsley, 1992, Parasitology Today 8: 69-
71) was used for the inspection. The inspection of the thin layer smear sample after AO staining was performed at 400 times magnification with an optical microscope and halogen lamp (MDM-K-DC; Tokyo Optics) equipped with an interference filter for exciting AO dye. It was done using. 2-3 ml of venous blood was collected from a malaria-positive patient over 6 years old. Coagulated blood or heparinized blood was sent to Japan in the cold, with an additional thin layer and thick smear. 1-3: Isolation of Plasmodium DNA A known method for preparing a protozoan DNA template was prepared from a methanol-fixed concentrated smear (Kawamoto et al., 1996, Journal.
of Clinical Microbiology 34: 2287-2289; Liu et a
l., 1998, Journal of Clinical Microbiology 36: 337
8-3381; Zhou et al., 1998, Tropical Medicine and In
ternational Health 3: 304-312) and from the blood DNA Isolation Kit (High Pure PCR Templete Preparatio
n Kit, Boeringer Mannheim GmBH). 1-4: Molecular diagnosis using full-nested PCR and microtiter plate hybridization (MPH) Full-nested PCR diagnosis modified from semi-nested PCR (Kimura et al., 1997, Parasitology International
46: 91-95) to the block 9 region of the SSU rRNA gene (Qari
et al., 1994, Gene 150: 43-49), using universal primers P1F-Up and P1R (see FIG. 2). PCR amplification uses AmpliTaq gold polymerase
(PE Applied Biosystems), 96 ° C for 10 minutes, 94
36 cycles of 30 seconds at 55 ° C, 30 seconds at 55 ° C and 60 seconds at 72 ° C, followed by a final extension of 72 ° C for 8 minutes. The PCR product was electrophoresed on a 2.5% agarose gel to confirm amplification.

The primary PCR product was diluted 1: 100 in TE buffer, and this was used as a template for type diagnosis by secondary PCR. In the secondary PCR, four species (PrnR, PfR, P
Species-specific 3'primers prepared for vR and PoR2; boxed box in Figure 3) were used in combination with P1F. PoR2
Is a literature method (Kimura et al., 1997, Parasitology Inte
rnational 46: 91-95) was used. Secondary PCR was performed using AmpliTaq gold polymerase with the following cycle parameters: 94 ° C 10 minutes, 94 ° C 30 seconds 21
Cycle, 55 ° C 30 seconds and 72 ° C 60 seconds, then 72 ° C 8 minutes as final extension. Negative controls with water were run with samples in all experiments. False positives or cross reactions were absent, including the four human malaria species.

MPH diagnosis (Kawamoto et al, 1996, Journal
of Clinical Microbiology 34: 2287-2289; Liu et a
l., 1998, Journal of Clinical Microbiology 36: 337
8-3381; Kawamoto et al., 1999, Parasitology Today
15: 442-426), PCR was performed using 5'-biotinylated primers, MPH-1 and MPH-2 (see FIG. 3), under the same conditions as the initial PCR reaction in the nested PCR diagnosis. The PCR product was then incubated at 55 ° C for 1 hour in a microtiter plate (PCR-MPH plate, Wakunaga Pharmaceutical
4 species-specific probes immobilized in wells (see lower case in Figure 3; Pf, 5'-GTC ACC TCG AAA GAT G
AC TT-3 '; Pv, 5'-TAA ACT CCG AAG AGA AAA TTC-3'; P
m, 5'-ACT CAT ATA TAA GAA TGT CTC-3 '; Po, 5'-AAT T
TC CCC GAAAGG AAT TTT C-3 ') and a generic probe for the genus Plasmodium (5'-CGG CATAGT TTA TGG TTA AG-
3 ') was hybridized and used for colorimetric assay with alkaline phosphatase-conjugated streptavidin system. A405nm measurement was performed using a microplate reader (MPR-A4; Tosyo). 1-5: Target sequence of two PCR analyzes and whole sequence analysis of SSU rRNA gene The target sequence of 15 samples diagnosed positive for P. malariae was analyzed by the nested PCR method. Amplified DNA product using P1F and specific reverse (PmR) primer
TA clining system (Invitrogen) plasmid pCRII
Cloned in. Sequence analysis of the target fragment in 5 positive clones of each sample was performed on both DNAs.
For chains, Big-dye Termin using ABI310 Sequencer
ator Sequencing kit (PE Applied Biosystems) was used.

Six samples with only a single infection with P. malariae-like protozoa were used for the whole sequence analysis. PCR amplification was performed at 96 ° C. for 10 minutes, 94 ° C. for 30 seconds for 36 cycles, 55 ° C. for 60 seconds and 72 ° C. for 60 seconds, and then 72 ° C. for 10 minutes as the final extension. The primers used are: 18S F (5'-AAC CTG
GTT GAT CTT GCC AGT AGT-3 '), 18S F1 (5'-CGA TTCCG
G AGA GGG AGC CTG-3 '), and 18S F2 (5'-GGT AAT T
CC AGC TCC AAT AG-3 '), P1F-Up, PmR, 18S F3 (5'-TG
G ATG GTG ATG CAT GGC CCG T-3 ') and 18SR (5'-TAA
TGA CCT TCC GCA GGT TCA CC-3 '). The PCR product was cloned into the pCRII plasmid, clone 5 of each sample.
For each, sequence analysis of both DNA strands was performed using ABI377 Sequencer.

The obtained sequence was compared with the following sequences reported in the database:
Complete sequence of P. malariae Uganda-1 / CDC (M54897; Go
manet al., 1991, Molecular and Biochemical Parasit
ology 45: 281-288), derived from Greece (AF014942; Vinet
z et al., 1998, The New England Journal of Medicin
e 338: 367-371), Papua New Guinea (PNG; AF145)
336; Mehlotra et al., 2000, American Journal of Tr
opical Medicine and Hygiene 62: 225-231) Origin and origin unknown (London Sch. Trop. Med. Hyg strain; U78741;
Li et al., 1995, Experimental Parasitology 81: 182
-190) isolate partial sequence, and P. brasili
anum (AF130735; Fandeur et al., 2000, Parasitology
120: 11-21), and P. falciparum (M19172; McCutc
han et al., 1988, Molecular and Biochemical Parasi
tology 28: 63-68), P. vivax (X13926; Qari, Goldma
net al., 1994, Gene 150: 43-49) and P. ovale (L4
8987; Qari et al., 1996, Molecular Phylogenetic Ev
olution 6: 157-165) full sequence of the A gene. 1-6: Sequencing analysis of CS gene CS (sporozoite peripheral protein) gene analysis was performed using PmCS F (5'-ATG AAG AAG TTA TCT G
TC TTA-3 '), PmCS F1 (5'-GCT GTT GAA AAT AAA TTG A
A-3 '), PmCS F2 (5'-AAT AAA AGG ACA ATC AGG G-
3 '), PmCS F3 (5'-AAA GTG GAT GCA AAT ACG AA-
3 '), PmCS R (5'-TGA AAG AGT ATT AAG ACT AA-3') and PmCS R2 (5'-TTC GTA TTT GCA TCC ACT TT-3 '), 96 ° C 10 minutes, 94 ° C 30 36 cycles of seconds, 50 ° C. for 60 seconds and 72 ° C. for 60 seconds, followed by a final extension of 72 ° C. for 10 minutes. The PCR product was cloned into the pCRII plasmid,
ABI377 Seque for 5 clones of 8 samples each
Sequence analysis of both DNA strands was performed using ncer.

The obtained sequence was compared with the following sequences reported in the database. Ie Ug
anda-1 / CDC (J03992; Lal, de la Crutz, Campbell et
al., 1988, Molecular and Biochemical Parasitology 3
0: 291-294), China-1 / CDC (U09766l Qari, Collins e
t al., 1994, American Journal of Tropical Medicine
and Hygiene 50: 45-51), Cameroon and Ivory Coast (AJ001523-26 and AJ002576-83; Tahar et a
l., 1998, Molecular and Biochemical Parasitology 9
2: 71-78), and P. brasilianu.
m (J03203; Lal, de la Cruz, Collins et al., 1988,
Experimental Parasitology 81: 182-190). 2: Results 2-1: Morphological findings A total of 3,294 volunteers were investigated, and malaria parasite was detected in 1,416 of them. During a malaria survey in September 1998, the inventor of this application identified a unique form of P. malariae-like protozoa in three Dawei patients (DW166, 187, 330) and Myi.
9 patients with tkyina (MK101, 112, 140, 169, 180, 196, 2
14, 301, 470). The most prominent of the protozoan morphological features in all cases was the specific morphology of the nucleus and cytoplasm at the early trophozoite stage. These could be grouped into two morphological types, A and B.

Most of the nuclei of type A (FIG. 4A) are rod-shaped or dot-shaped, and are divided into 2-3 fragments (FIG. 4, A2). Cytoplasm is dense and typical of P. mal
Somewhat larger than the ariae ring (Fig. 4, A2-3). No typical ring morphology is found, as observed in four human malaria species. No expansion of infected red blood cells is seen. Giemsa staining (FIG. 4, A3) confirmed similar morphological characteristics described above. No Schuffner spots are observed. Overall, type A parasites are morphologically Emin (1914, B
ulletin de la Societe de Pathologie Exotique 7: 38
It is very similar to P. vv minuta reported by 5-387) (Fig. 4, A1).

In contrast, the cytoplasm of type B early trophozoites (FIG. 4B) is very thin, amoebic and atypical. The chromatin is generally rod-shaped, rod-shaped, or curved (Fig. 4, B2), and is hardly punctate. In many malaria parasites, the chromatin is also divided into two fragments. No expansion of infected red blood cells was observed, Schf
No fner spots are observed by Giemsa staining (Fig. 4, B3). Therefore, this type is Stephens (1914, Proceeding
s of the Royal Society of London (Series B) 87:37
5-377; 1915, Annals of Tropical Medicine and Paras
Itology 9: 169-172) and is similar to P. tenue (Fig. 4, B1).

Following these initial findings, similar P. malar
34 additional cases of iae-like parasite infection 1998
It was detected in the period from November to February 2000. These cases apply throughout Myanmar, namely the Mandalay Division (KM14
1, MM277, M18, 24, 29, 37,43, 74, 117, 123, 133, 1
38, 145, 151, 163, 168, 180, 183, 196, 199, 328, 3
33, 359, 369), Thaton (TA138), Dawei (DW199, 20)
0), Muse (MS1) and Than Dwe (TD60, 120, 145, 19
5, 205, 234). In some of these cases, two malaria parasite infections were observed in one red blood cell (Fig. 4, A4, B4), which was normal P. malariae.
It has different characteristics than infection, however, Emin (1914)
And Stephens (1914, 1915; see FIG. 4, B1). Microscopically 5 out of 46 cases (DW166, MK
140, KM141, M18, TD60) seemed to be solely infected with P. malariae-like protozoa. Typical P. malariae band morphology, schizonts and / or gametocysts were also observed in these smears, suggesting that protozoa with this specific morphology may belong to the P. malariae group, and early Trophozoite stage only typical P. mal
It may be different from the morphology of ariae. P.
Upon mixed infection with falciparum and / or P. vivax, both P. falciparum and P. vivax parasites displayed normal morphological characteristics (unpublished data). 2-2: PCR diagnosis All 46 samples suspected of being infected with P. malariae-like protozoa were confirmed by full-nested PCR diagnosis.
It was confirmed to be positive. 9 cases (DW166, 330, MK14
0, 180, KM141, M18, TD60, 195, 234) is P. malariae
Although it was a single infection, the other 37 cases were P. falciparum and P. vi.
Co-infection with vax, P. ovale, or a combination of these. Mono-infection was more frequent in the newer type compared to conventional P. malariae infection. About 28 out of 46 cases, gel electrophoresis was performed on a 2.5% agarose gel using the nested PCR product.
A 4-5 bp shorter fragment appeared as compared to the P. malariae positive sample. In addition, three samples (DW330, MK140, MK470) had two bands of different sizes, one normal and one short. These results were confirmed on a 4% long agarose gel.

Other PCR diagnostics for four human malaria species,
That is, in the microtiter plate hybridization (MPH) assay, only 28 samples with relatively short PCR products were all P. malariae negative. Three of these samples (DW166, MK180, KM141) did not hybridize with the four human malaria probes and reacted only with the general probe of the genus Plasmodium. Another 18 samples (DW166, MK140 and MK470
(Including) was MPH-positive for P. malariae. These results suggested that 28 samples had a sequence mutation in the probe region used for MPH diagnosis. 2-3: Sequence analysis of PCR products in the target region using two types of PCR diagnosis. Sequence analysis of the target region used in PCR diagnosis was performed using P. mala.
9 samples of riae-like protozoa alone were infected, 3 of which produced two bands of different size by gel electrophoresis, the other 3 were randomly selected samples . The sequences obtained from both DNA strands in all samples were identical in 5 clones in each sample and two sequence types were found (Fig. 3). Type 1 was all derived from MPH negative samples (N = 5, DW166, 187, MK18
0, KM141, TD145), and a P. malariae Uganda-1 strain sequence has a 4 bp deletion (TTAT) in the probe region. Type 2 is all MPH
Derived from positive samples (N = 7, TA138, M18, TD60, 120,
195, 205, 234), which also has a sequence different from that of Uganda-1 and has one base substitution (ATA) in the probe region.
T) is seen. The 4bp deletion was previously investigated by the inventors in Vietnam (Kimura et al., 1997, Parasitology In.
ternational 46: 91-95), which is identical to that found in P. malariae isolates, while other sequence mutations were isolated from other Vietnamese isolates (SB61), from Papua New Guinea. It was found to be identical to that found in one of the detached bodies, P. brasilianum (Fig. 3). DW
The 330, MK140 and MK470 contain both type 1 and type 2 sequences, which is presumably the appearance of two different size bands on gel electrophoresis in these samples. From the above results, these malaria parasites are known at the molecular level, and are known to be known as P. falciparum and P.
It was clearly confirmed to be independent of the morphology and the isolate. In the morphological context, type 1 sequences are associated with type A protozoa (P.
uta-like) use case, while the type 2 sequence has a type B protozoa (P.
tenue) appears in the use case. Below, type 1 (type A)
Type 2 and Type B (P. minuta) and P. te, respectively
Described as nue-like protozoa. 2-4: Sequence analysis of SSU rRNA gene The entire sequence analysis of SSU rRNA gene was performed on single infected sample 6
We did about each (P.minuta: DW166, MK180, KM14
1; P. tenue: M18, TD195, 234). Sequence analysis on both DNA strands was identical in the 6 clones in each sample, reaffirming that these protozoa belonged to the P. malariae group (Fig. 1). Between P. minuta and P. tenue-like protozoa, only two regions (5 bp in total) differ in their respective sequences. Uganda-1
27 and 28 regions, respectively, differ when compared to the sequence. However, 12 of these regions are conservative or semi-conservative in the other three species, which is why Uganda
-1 suggests that the original sequence may have been misread. In the P. malariae-specific sequence, the new type has three other P. malariae isolates and P. brasili at the positions shown in FIG.
Different from any of the anum. Taken together, 2 examples of PC
P. mal containing the target region (9th block) at R diagnosis
ariae (Uganda-1 and Papua New Guinea isolates)
And P. m. For reported sequences for P. brasilianum.
inuta-like protozoa has a 4bp deletion (TTAT) that is different from the others,
P. tenue-like protozoa is P. malariae against P. brasilianum
Closer relationship than Papua New Guinea (7 bp difference) or Uganda-1 isolates. 2-5: Analysis of CS gene P. malariae (Lal, de la Cruz, Campbell et al., 198
8; Qari, Collins et al., 1994; Tahar et al., 1998)
And P. brasilianum (Lal, de la Cruz, Collins et
al., 1988) has three regions, a pre-repetition region, four amino acids (NED in NDAG / NAAG and China-1).
It is composed of 49-61 repeat regions (1 copy of G) and a post-repeat region. Eight samples were selected for analysis of the CS gene, five of which were single infected (DW166, MK18
0, TD60, 195, 234), 3 co-infected with P. vivax (TD
120, 145, 205).

In the pre-repeat region, the test sample is Uganda
-1, China-1, and sub-Saharan isolates. However, the start of the repeat region is different and DW16
NDEG NDAG N for 6 and MK180 (both P. minuta-like protozoa)
DAG (China-1 and P. brasilianum), TD145 (P. minu
ta-like protozoa), NDAG NDAG NDAG (6 isolates from Uganda-1 and Cameroon), and TD60, 120, 195, 205, 23
4 (all P. tenue-like protozoa) NDAG NDAG NAAG (6 isolates from Cameroon and isolates from Côte d'Ivoire)
Met.

In the repeat region, P. malariae isolates and
A polymorphism with repeat length as reported in P. brasilianum was also observed (Fig. 5). 3 samples (DW1
66, MK180, TD145, all P. minuta-like protozoa) had 51 repeat units identical to Uganda-1, but 5 samples (TD60, 120, 195, 205, 234, all tenue-like protozoa) The sequence length was about 96, 108 and 132 bp (59, 60 and 62 repeats, respectively).

In the post-repeat region, the same sequence was present in each new strain, and two non-silent nucleic acid mutations were found. 3
At amino acid 35, Uganda-1 and P. brasilianum
E is mutated to G, which is observed in China-1 and Cameroon 402. At amino acid 382, G in Uganda-1 was changed to D, which is the same as that reported in many other P. malariae isolates and P. brasilianum.

[0046]

INDUSTRIAL APPLICABILITY As described in detail above, the invention of this application provides two completely new types of Plasmodium vivax strains with their respective SSU rRNA sequences. This makes it possible to further improve the accuracy of malaria diagnosis by DNA diagnosis or the like.

[0047]

[Sequence list]                                SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> New strains of Plasmodium marariae and malaria       diagnosis <130> NP01426-YS <140> <141> <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 2153 <212> DNA <213> Plasmodium malariae <400> 1 aacctggttg atcttgccag tagtcatatg cttgtctcaa agattaagcc atgcaagtga 60 aagtatatgc atattttata tgtagaaact gcgaacggct cattaaaaca gttatagtct 120 acttgacatt ttttttataa ggataactac ggaaaagctg tagctaatac ttgctttaat 180 actcttaatt ctttatgttt tttgagtatg tatttgttaa gccttataag agaaaagttt 240 attaacttaa ggaattataa caaagaagta acacataata aatttcgatt tatttagtgg 300 tatcaatcga gtttctgacc tatcagcttt tgatgttagg gtattggcct aacatggcta 360 tgacgggtaa cggggaatta gagttcgatt ccggagaggg agcctgagaa atagctacca 420 catctaagga aggcagcagg cgcgtaaatt acccaattct aaagaagaga ggtagtgaca 480 agaaataaca atgcaaggcc aaattttggt tttgcaattg gaatgatggg aatttaaaac 540 cttcccagaa ggcaattgga gggcaagtct ggtgccagca gccgcggtaa ttccagctcc 600 aatagcgtat attaaaattg ttgcagttaa aacgctcgta gttgaatttc aaggaatcaa 660 tattttaagt aatgctttgt atatttataa caaagttgta cattaagaat aaacgccaag 720 cgttatattt tttctgttac attttgtttt attaatatat atatgcgttc ttattataaa 780 aatgattctt tttaaaattc ttttgtataa ttttttatgc atgggaattt tgttactttg 840 agtaaattag agtgttcaaa gcaaacagtt aaaacagttt ctgtgtgtttga atactacagc 900 atggaataac aaaattgaac aagtcagaat tttgttcttt tttcttattt tggcttagtt 960 acgattaata ggagtagctt gggggcattt gtattcagat gtcagaggtg aaattcttag 1020 attttctgga gacaagcaac tgcgaaagca tttgcctaaa atacttccat taatcaagaa 1080 cgaaagttaa gggagtgaag acgatcagat accgtcgtaa tcttaaccat aaactatgcc 1140 gactaggtgt tggatgatag agtaaaaaat aaaagagaca ttcatatgag tgtttctttt 1200 agatagcttc cttcagtacc ttatgagaaa tcaaagtctt tgggttctgg ggcgagtatt 1260 cgcgcaagcg agaaagttaa aagaattgac ggaagggcac caccaggcgt ggagcttgcg 1320 gcttaatttg actcaacacg gggaaactca ctagtttaag acaagagtag gattgacaga 1380 ttaatagctc tttcttgatt tcttggatgg tgatgcatgg ccgtttttag ttcgtgaata 1440 tgatttgtct ggttaattcc gataacgaac gagatcttaa cctgctaatt agcggtaaat 1500 acactatatt cttaagtgaa attagaatat agataaattg tgctaatttt gattaaaata 1560 ttagaatgtt ttttttaata aaaacgttct tttccctttt tttcttaatt atgcatattt 1620 attctttttc ttttttcgca taagaatgta tttgcttaat tgtaaagctt cttagaggaa 1680 cgatgtgtgt ctaacacaag gaagtttaag gcaacaacag gtctgtgatg tccttagatg 1740 aactaggctg cacgcgtgct acactgatat gtataacgag tatttaaaaa tatatatctt 1800 gttatgttat atgtatttct atatgtatgc atgcaaagaa tatatagttt tcctccactg 1860 aaaagtgtag gtaatctttt tcaatacata tcgtgatggg gatagattat tgcaattatt 1920 aatcttgaac gaggaatgcc tagtaagcat gattcatcag attgtgctga ctacgtccct 1980 gccctttgta cacaccgccc gtcgctccta ccgattgaaa gatatgatga attgtttgga 2040 caaggaaaaa gggtttttat tcttttttct ggaaaaatcg taaatcctat cttttaaagg 2100 aaggagaaag tcgtaacaag gtttccgtcg gtgaacctgc ggaaggatca tta 2153 <210> 2 <211> 2158 <212> DNA <213> Plasmodium malariae <400> 2 aacctggttg atcttgccag tagtcatatg cttgtctcaa agattaagcc atgcaagtga 60 aagtatatgc atattttata tgtagaaact gcgaacggct cattaaaaca gttatagtct 120 acttgacatt ttttttataa ggataactac ggaaaagctg tagctaatac ttgctttaat 180 actcttaatt ctttatgttt tttgagtatg tatttgttaa gccttataag agaaaagttt 240 attaacttaa ggaattataa caaagaagta acacataata aatttcgatt tatttagtgt 300 gtatcaatcg agtttctgac ctatcagctt ttgatgttag ggtattggcc taacatggct 360 atgacgggta acggggaatt agagttcgat tccggagagg gagcctgaga aatagctacc 420 acatctaagg aaggcagcag gcgcgtaaat tacccaattc taaagaagag aggtagtgac 480 aagaaataac aatgcaaggc caaattttgg ttttgcaatt ggaatgatgg gaatttaaaa 540 ccttcccaga aggcaattgg agggcaagtc tggtgccagc agccgcggta attccagctc 600 caatagcgta tattaaaatt gttgcagtta aaacgctcgt agttgaattt caaggaatca 660 atattttaag taatgctttg tatatttata acaaagttgt acattaagaa taaacgccaa 720 gcgttatatt ttttctgtta cattttgttt tattaatata tatatgcgtt cttattataa 780 aaatgattct ttttaaaatt cttttgtgta attttttatg catgggaatt ttgttacttt 840 gagtaaatta gagtgttcaa agcaaacagt taaaacagtt tctgtgtttg aatactacag 900 catggaataa caaaattgaa caagtcagaa ttttgttctt ttttcttatt ttggcttagt 960 tacgattaat aggagtagct tgggggcatt tgtattcaga tgtcagaggt gaaattctta 1020 gattttctgg agacaagcaa ctgcgaaagc atttgcctaa aatacttcca ttaatcaaga 1080 acgaaagtta agggagtgaa gacgatcaga taccgtcgta atcttaacca taaactatgc 1140 cgactaggtg ttggatgata gagtaaaaaa taaaagagac attcatatat atgagtgttt 1200 cttttagata gcttccttca gtaccttatg agaaatcaaa gtctttgggt tctggggcga 1260 gtattcgcgc aagcgagaaa gttaaaagaa ttgacggaag ggcaccacca ggcgtggagc 1320 ttgcggctta atttgactca acacggggaa actcactagt ttaagacaag agtaggattg 1380 acagattaat agctctttct tgatttcttg gatggtgatg catggccgtt tttagttcgt 1440 gaatatgatt tgtctggtta attccgataa cgaacgagat cttaacctgc taattagcgg 1500 taaatacact atattcttaa gtgaaattag aatatagata aattgtgcta attttgatta 1560 aaatattaga atgttttttt taataaaaac gttcttttcc ctttttttct taattatgca 1620 tatttattct ttttcttttt tcgcataaga atgtatttgc ttaattgtaa agcttcttag 1680 aggaacgatg tgtgtctaac acaaggaagt ttaaggcaac aacaggtctg tgatgtcctt 1740 agatgaacta ggctgcacgc gtgctacact gatatgtata acgagtattt aaaaatatat 1800 atcttgttat gttatatgta tttctatatg tatgcatgca aagaatatat agttttcctc 1860 cactgaaaag tgtaggtaat ctttttcaat acatatcgtg atggggatag attattgcaa 1920 ttattaatct tgaacgagga atgcctagta agcatgattc atcagattgt gctgactacg 1980 tccctgccct ttgtacacac cgcccgtcgc tcctaccgat tgaaagatat gatgaattgt 2040 ttggacaagg aaaaagggtt tttattcttt tttctggaaa aatcgtaaat cctatctttt 2100 aaaggaagga gaaagtcgta acaaggtttc cgtcggtgaa cctgcggaag gatcatta 2158

[Brief description of drawings]

[Figure 1] Plasmodium malariae (Pm), P. brasilianum
(Pb) Different sequences in the SSU rRNA genes of two new types and four human malaria species. The number of nucleotides indicates the position within the Uganda-1 sequence. Bold letters indicate different nucleotides in each species. P. malariae isolates include Uganda-1, Greece, London Sch. Trop. Med. H
Includes yg strain (LSTMH), and Papua New Guinea (PNG). Pf; P. falciparum; Pv, P. vivax; Po, P. oval
e.

FIG. 2 shows known sequences of Plasmodium vivax and human malaria diagnostic probe sequences that are commercially available in Japan (lowercase letters).

Figure 3: SSU rRNA used for nested PCR and MPH diagnosis
The target sequence of the gene. Sequence is Plasmodium malariae (Pm) U
Shown is the sequence of the ganda-1 strain and a partial sequence of the Papua New Guinea (PNG) strain and P. brasilianum (Pb). New model 1 and new model 2 are P. vv minuta
Shows two new types corresponding to P. tenue and P. tenue-like protozoa. SB41 and SB61 are strains derived from Vietnam (Kawamoto et al., 1996, Journal of Clinical Microb
Biology 34: 2287-2289; Kimura et al., 1997, Parasit
ology International 46: 91-95). The boxed sequence is the target of the species-specific 3'-primers (PmR, PfR, PvR and PoR2) used for nested PCR. The lower case part indicates the species-specific probe region as well as the general Plasmodium probe used for MPH diagnosis. Pf; P. falcipa
rum; Pv, P. vivax; Po, P. ovale.

FIG. 4 Emin and Stephens protozoa and type A (Plasm
odium vivax, variety minuta) and B type (P. tenue
) Observation of early trophozoites of protozoa by acridine orange (AO) and Giemsa staining. 6 bars
μM is shown. A1: P. vvminuta early trophozoites, drawn by Emin (1914). A2: Type A protozoa (DW166)
AO staining; folds are very early in trophozoites (MK18
0). A3: Giemsa stain of A-type protozoa (DW166). A4: AO staining of two protozoa A (KM141) in red blood cells. B1: P. tenue's early trophozoites, drawn by Stephens (1914). B2
-3: AO and Giemsa staining of B-type protozoa (DW330 and MK214, respectively). B4: AO of two type B protozoa (M29) in red blood cells
staining.

FIG. 5: Polymorphism of repeat length in the CS gene repeat region in a novel Plasmodium vivax parasite. PCR products of TD60 and 120 (both P. tenue-like protozoa) are TD145, DW166 and M
The sequence is longer than that of K180 (all P. minuta-like protozoa).

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/569 C12N 15/00 ZNAA // C12M 1/00 FF term (reference) 4B024 AA13 CA01 HA12 HA15 4B029 AA07 AA23 CC03 FA03 4B063 QA18 QA19 QQ02 QQ05 QQ42 QQ79 QR08 QR32 QR42 QR48 QR55 QR62 QS25 QS33 QS34 QX01 4B065 AA86X AA86Y AC20 CA24 CA25 CA46 4H045 AA11 BA10 CA22 DA76 EA52 FA74

Claims (9)

[Claims] 1. The nucleotide sequence of the SSU rRNA gene is SEQ ID NO: 1.
Is a Plasmodium vivax. 2. The nucleotide sequence of the SSU rRNA gene is SEQ ID NO: 2.
Is a Plasmodium vivax. 3. A purified polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1. 4. A purified polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2. 5. An oligonucleotide that hybridizes to the purified polynucleotide of claim 3 or 4. 6. The oligonucleotide according to claim 5, which is bound with a labeling substance. 7. A microarray comprising the purified polynucleotide according to claim 3 or 4, or the oligonucleotide according to claim 5. 8. An antibody against Plasmodium vivax according to claim 1 or 2. 9. A method for diagnosing malaria, which comprises detecting the presence of Plasmodium vivax according to claim 1 or claim 2 in a test sample.