TECHNICAL FIELD

The invention relates to a method for the preparation of a research toolkit comprising Leishmania amastigotes and promastigotes to be used in research for the diagnosis, medication, and vaccination of diseases caused by Leishmania parasites. The toolkit of the invention also contains Leishmania genomic DNA.

BACKGROUND

The whiplash parasites that live in human blood are of the genus Leishmania and the genus Trypanosoma from the family Trypanosomatidae. They cause disease in humans and other vertebrates. Approximately 12 million people in 98 countries worldwide suffer from cutaneous leishmaniasis and visceral leishmaniasis caused by parasitic diseases and transmitted by Phlebotomus, and 350 million people are at risk. It is estimated that 2 million new cases are added to these figures every year, approximately one and a half million of these cases are cutaneous leishmaniasis and half a million are visceral leishmaniasis. It is known that the annual number of deaths due to these parasites is 60 thousand, and it is estimated that approximately every 20 seconds, one person has cutaneous leishmaniasis. The gold standard method in direct diagnosis is the appearance of amastigotes in the clinical material and the production of promastigotes in the NNN medium.

Leishmania species pass on to humans and other vertebrates during the blood-sucking of Phlebotomus (Tartar flies). In their evolution, they require 2 separate mansions, vertebrate and invertebrate. They are found in the form of Leishmania in the vertebrate host and Leptomonas in the invertebrate host. The form of leishmania is called amastigote, and the form of leptomonas is called promastigote.

There are more than 20 species of Leishmania species, mainly Leishmania major, donovani, tropica, aethiopica, infantum, braziliensis. These may be in dogs and humans, they are zoonotic. L. tropica is a causative agent of cutaneous leishmaniasis. It is also known as the orchestral boil. Recently, L. major species have been isolated from patients with cutaneous leishmaniasis obtained from humans and species have been determined molecularly. In the state of the art, there are many methods, preparations, or toolkits for the diagnosis of the mentioned whips. However, there is no basic research toolkit in which all three (promastigote, amastigote, and genomic DNA) are together to form the basis for the studies to be carried out for diagnosis, medicine, and vaccine.

The patent application EP0716697B1 relates to obtaining a gene from Leishmania in the form of an amastigote. The use of the obtained Leishmania genes and proteins in vaccine form as means for the production of immunological reagents and the production of attenuated variants of Leishmania has been described.

Patent application US6375955B1 relates to antigens in the diagnosis and treatment of Leishmania. Compositions and methods for the treatment, prevention, and diagnosis of Leishmania are described.

The present invention, on the other hand, provides the basic material required for the diagnosis, drug, and vaccine methods of the diseases caused by the Leishmania parasites, which are in the state of the art, and provides an easy-to-implement, low-cost method for a high-sensitivity research toolkit containing the amastigotes and promastigotes of the Leishmania and their genomic DNA to the scientists who will conduct a research on this subject.

BRIEF DESCRIPTION OF THE INVENTION

The main object of the invention is to present a method for preparing a research toolkit containing Leishmania amastigotes and promastigotes to be used in the research to be used for the diagnosis, medication, and vaccination of diseases caused by Leishmania parasites.

The toolkit of the invention is used especially in the diagnosis, treatment, and/or vaccine studies of diseases caused by Leishmania parasites.

In one embodiment, the toolkit of the invention also comprises the genomic DNA of the parasite.

According to an embodiment of the present invention, said Leishmania parasite is selected from a list comprising Leishmania tropica, Leishmania major, Leishmania infantum, Leishmania donovani, and Leishmania aethiopica. According to one embodiment of the invention, the method of the invention comprises the following steps: a. taking said parasites into separate tubes after preparing the promastigote and amastigote suspensions separately, b. adding preservatives up to 15% of their volume on the substances prepared in step a, c. distributing the mixtures obtained in step b to 1 ml in separate tubes, d. preserving the tubes overnight at -80 to -90°C, e. transferring the preserved tubes to the liquid nitrogen tank, f. bringing together tubes containing live Leishmania amastigotes and promastigotes.

In this embodiment of the present invention, the suspensions in step a of the method of the invention are prepared to be 10 ⁷ - 10 ⁸ parasites/ml.

In an embodiment of the present invention, dimethyl sulfoxide (DMSO) is added as a preservative in step b.

In an embodiment of the present invention, the tubes in the method of the invention are cryotubes.

In this embodiment of the present invention, the holding process in step d of the method of the invention is carried out at -86°C for 24 hours.

In this embodiment of the invention, said method further comprises the following steps: i. isolating genomic DNA, ii. after adjusting the purity value of the DNA obtained in step i to 260/280 value 1 .7 - 2 and the concentration value to 10 pg, taking it into tubes to be 100 pl, iii. adding the tube containing the isolated DNA to the research toolkit of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The main object of the invention is to present a method for the preparation of a research toolkit containing Leishmania amastigotes and promastigotes to be used in the research to be used for the diagnosis, medication, and vaccination of diseases caused by Leishmania parasites. In one embodiment, the toolkit of the invention also comprises the genomic DNA of said Leishmania parasite. Genomic DNA isolation was performed with Qiagen DNeasy Blood & Tissue Kit. In the studies to be used, the ideal working purity value of DNA was transferred to the tubes as 100 pl after adjusting the concentration value to 10 pg with 260/280 value 1.7-2 measured using Nanodrop spectrophotometer device.

According to an embodiment of the present invention, said Leishmania parasite is selected from a list comprising Leishmania tropica, Leishmania major, Leishmania infantum, Leishmania donovani, and Leishmania aethiopica.

Early diagnosis toolkits, medication, and vaccines are of vital importance in diseases caused by the parasite family mentioned in the invention. For this reason, there is a need for rapid and new diagnostic toolkits, drugs, and vaccines related to the disease. The method of the present invention has been carried out with laboratory equipment that is readily available and accessible to those skilled in the art. Therefore, the method is both easy to apply and low cost. Thus, a high-precision toolkit used for the diagnosis of diseases caused by Leishmania parasites, an effective vaccine, and a low-cost, easy-to-use method for preparing a research toolkit to pass the disease on.

According to one embodiment of the invention, the method of the invention comprises the following steps: a. taking said parasites into separate tubes after preparing the promastigote and amastigote suspensions separately, b. adding preservatives up to 15% of their volume on the substances prepared in step a, c. distributing the mixtures obtained in step b to 1 ml in separate tubes, d. preserving the tubes overnight at -80 to -90°C, e. transferring the preserved tubes to the liquid nitrogen tank, f. bringing together tubes containing live Leishmania amastigotes and promastigotes.

In this embodiment of the present invention, the suspensions in step a of the method of the invention are prepared to be 10 ⁷ - 10 ⁸ parasites/ml.

In one embodiment of the present invention, dimethyl sulfoxide (DMSO) is added as a preservative in step b. A homogeneous mixture should be obtained with the preservative used in this step to prevent any phase formation and the desired homogeneous mixture is achieved with the dimethyl sulfoxide used in the present invention.

In an embodiment of the present invention, the tubes in the method of the invention are cryotubes.

In this embodiment of the present invention, the holding process in step d of the method of the invention is carried out at -86°C for 24 hours. After this process, the cryopreservation process of the substances transferred to the liquid nitrogen tank is completed. The toolkits of the present invention are obtained by combining the live Leishmania amastigotes and promastigotes obtained in separate tubes as a result of cryopreservation.

In this embodiment of the invention, said method further comprises the following steps: i. isolating genomic DNA, ii. after adjusting the ideal working purity value of DNA obtained in step i in the studies to be used is 260/280 value 1.7 - 2 measured using a Nanodrop spectrophotometer device and the concentration value to 10 pg, taking it into tubes to be 100 pl, iii. adding the tube containing the isolated DNA to the toolkit of the invention.

Genomic DNA from Leishmania reference strains is available for purchase from a variety of companies. However, these commercially available strains have been isolated for as long as 20-30 years, and many of them lose their genetic characteristics and virulence. Erroneous results are obtained if these isolates are used in the diagnosis of diseases caused by Leishmania parasites. Thanks to the genomic DNA added to the research toolkit in the method of the present invention, the sensitivity and reliability of the research toolkit increase.

Example 1 - Toolkit containing Leishmania live forms of Promastigote and Amastigote

Preparation of Leishmania live Promastigote form

Promastigote suspensions were obtained from Leishmania tropica, Leishmania major, Leishmania infantum, Leishmania donovani, and Leishmania aethiopica isolates stored in a liquid nitrogen tank in the parasite bank to be 10 ⁸ parasites/ml. The mixture was homogenized by adding 15% of its volume of DMSO to each suspension obtained. Then, each mixture was distributed in sterile cryotubes with internal caps to be 1 ml. The cryotubes containing the mixtures were taken into cool cell boxes and the boxes were placed in a -86°C deep freezer. The boxes kept here overnight were removed from the freezer the next day and the cryotubes in the boxes were transferred to the liquid nitrogen tank and the cryopreservation process was completed.

Preparation of Leishmania live Amastigote form

Amastigote suspensions were obtained from Leishmania tropica, Leishmania major, Leishmania infantum, Leishmania donovani, and Leishmania aethiopica isolates stored in a liquid nitrogen tank in the parasite bank to be 10 ⁸ parasites/ml. The mixture was homogenized by adding 15% of its volume of DMSO to each suspension obtained. Then, each mixture was distributed in sterile cryotubes with internal caps to be 1 ml. The cryotubes containing the mixtures were taken into cool cell boxes and the boxes were placed in a -86°C deep freezer. The boxes kept here overnight were removed from the freezer the next day and the cryotubes in the boxes were transferred to the liquid nitrogen tank and the cryopreservation process was completed.

The research toolkit of the invention was obtained by combining the tubes containing the Leishmania live Promastigote and Amastigote forms prepared above.

Example 2 - Toolkit containing Leishmania viable forms of Promastigote and Amastigote and Leishmania Genomic DNA

Preparation of Leishmania genomic DNA

The Promastigote suspensions described in Example 1 were subjected to DNA isolation. DNA isolation process was performed with Qiagen DNeasy Blood & Tissue Kit. The value of the DNA obtained was checked with the help of a nanodrop device. After adjusting the purity value of DNA obtained in step i in the studies to be used is 260/280 value 1.7 - 2 measured using a Nanodrop spectrophotometer device and the concentration value to 10 pg, taking it into tubes to be 100 pl,

The research toolkit of the invention was obtained by combining the Leishmania live Promastigote and Amastigote forms and the tubes containing the genomic DNA in Example 1.

The protection scope of the invention is specified in the appended claims and cannot be strictly limited to those explained in this detailed description for illustrative purposes. It is evident that a person skilled in the art may exhibit similar embodiments in light of the foregoing without departing from the main theme of the invention.

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