Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-01-07
2000-02-08
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 2432, 536 2433, 536 237, C12Q 168, C12P 1934, C07H 2104
Patent
active
060226900
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to DNA specific to leishmania protozoans, and a method for detecting the leishmania protozoan by use of the DNA.
BACKGROUND ART
Leishmaniasis is a disease caused by infection with a protozoan of Leishmania genus (see, for example, IKAGAKU DAIJITEN 49, ENCYCLOPEDIA OF MEDICAL SCIENCES, published in April 1993, Kodansha). More than 12,000,000 people are estimated to be suffering from this disease. Leishmaniasis is one of the important diseases designated by WHO. The leishmania protozoans are classified morphologically into two types: amastigote and promastigote. The both types of protozoans are known to promulgate as the promasitgote type when the protozoan is transplanted to an appropriate culture medium.
Three species are known to infect humans: Leishmania donovani, Leishmania tropica, and Leishmania braziliensis.
These three species have not been differentiated from each other morphologically. However, in recent years, these species have come to be known to include subspecies and varieties by immunological and biochemical techniques.
The methods for detection of the pathogen include direct detection of the protozoan, and indirect estimation of infection with the protozoan by immune reaction or a like method.
Of these, the direct detection is sure, but requires long time and complicated operations, being not practicable in a short time and in large numbers.
The known indirect detection methods include Chopra's antimony reaction, Napier's aldehyde reaction, Branchari's euglobulin reaction, and so forth.
Further, complement-fixation reaction, indirect hemagglutination reaction, and indirect immunofluorometry are known as the immunological methods therefor.
The definite identification of the species is conducted by using cultivated protozoan by Adler's test, measurement of buoyant-density of kineplast DNA, isozyme analysis, and so forth as well as the immune reaction.
DISCLOSURE OF INVENTION
As explained above, the known methods of detection of the leishmania protozoan are not practically useful in view of precision, sensitivity, and operation efficiency, and are not applicable to development of a diagnosis medicine and a diagnosis kit for group examination and the like which are potent means for inhibiting the leishmaniasis infection, disadvantageously.
Moreover, although the decisive identification of the species is important for selection or establishment of treatment for the infection, no practically useful identification method is found.
With the above problems, the object of the present invention is to provide a sure and quick method for judging leishmania infection and identifying the species thereof in a large number, and to make possible the development of diagnosis medicines and diagnosis kits for group examination which is effective in inhibiting leishmania infection.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1A shows electrophoresis patterns of DNA amplified by a PCR method employing an LS-3 primer.
FIG. 1B shows electrophoresis patterns of DNA amplified by a PCR method employing an LS-3 primer.
FIG. 2A shows that amplification of DNAs of the F4 band is observed in all kinds of leishmanias when F4 detection primers (SEQ ID NO:5 and 6) are employed.
FIG. 2B shows that amplification of DNAS of the F4 band is observed in all kinds of leishmanias when F4 detection primers (SEQ ID NO:5 and 6) are employed.
FIG. 3A shows that amplification of DNAs of the F10 band is observed only in Leishmania braziliensis type when F10 detection primers (SEQ ID NO:2 and 3) are employed.
FIG. 3B shows that amplification of DNAs of the F10 band is observed only in Leishmania braziliensis type when F10 detection primers (SEQ ID NO:2 and 3) are employed.
FIG. 4 shows that a trypanosome, a relative parasite, gives no amplified DNA when the F4 detection primers and the F10 detection primers are employed.
BEST MODE FOR CARRING OUT THE INVENTION
The present invention is described below in detail by reference to the annexed drawings.
The present invention provides methods for
REFERENCES:
Maarten H.L. et al., Diagnosis of New World Leishmaniasis: Specific Detection of Species of the Leishmania Braziliensis Complex by Amplification of Kinetoplast DNA, Acta Tropica, (1992), vol. 52, 45-58.
Eresh S. et al., Identification and Diagnosis of Leishmania Mexicana Complex Isolates by Polymerase Chain Reaction, Parasitology, (1992), vol. 109, p. 423-433.
Smyth A.J. et al, Rapid and Sensitive Detection of Leishmania Kinetoplast DNA from Spleen and Blood Samples of Kala-Azar Patients, Parasitology, (1992) vol. 105, p. 183-192.
Piarroux R. et al., Isolation and Characterization of a Repetitive DNA Sequence from Leishamania Infantum: Development of a Visceral Leishmaniasis Polymerase Chain Reaction, Am. J. Trop. Med., (1993), vol. 49, p. 364-369.
Ikagaku Daijiten 49, Encyclopedia of Medical Seciences, Published in Apr. 1993, Kodansha (with concise English explanation).
Mimori T. et al., Classification of Leishmania Parasites, using kDNA Finger Printing Method by . . . , Japanese Journal of Parasitololy, Apr. 5-6, 1995.
Kaluza. GenBank Accession No. S72847, Jan. 1993.
Berbee et al. GenBank Accession No. U00977, Aug. 1993.
Weissenbach. Genbank Accession No. Z23401, Nov. 1994.
Kano. GenBank Accession No. D12624, Jan. 1994.
Mimori Tatsuyuki
Nakata Motomi
Sasaki Jiichiro
Saya Hideyuki
Myers Carla J.
Sumitomo Electric Industries Ltd.
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