Species identification, disease detection, and pathogenicity determination methods in eukaryotic exosomes and bacterial outer membrane vesicles.

Abstract

The invention relates to a method (100) that enables species determination, disease and pathogenicity detection in eukaryotic exosomes and bacterial outer membrane vesicles using isothermal nucleic acid amplification methods.

Description

1 TARIFF EUKARYUTIC EXOSOMES AND BACTERIAL OUTER MEMBRANE SPECIES IDENTIFICATION, DISEASE DETECTION AND IN VESCULAR EXAMPLES. PATHOGENICITY DETECTION METHOD Technical Area The invention relates to isothermal nucleic acid amplification methods using eukaryotic exosomes and Species identification in bacterial outer membrane vesicles, disease and pathogenicity. It relates to a method that enables the detection to be carried out. 10 Previous Technique Bacterial outer membrane vesicles (BDMVs) and eukaryotic exosomes (SPCs) are 15 cells formed as a result of different stress and disease factors in living cells. Budding produces lipids, 30-200 nm in size, formed by the cell. These are vesicles. During membrane formation, these vesicles are composed of different cells. It also includes elements such as proteins and nucleic acids. and many other small molecules that can give insight into the cell's internal environment. It carries molecules. Nucleic acids are found in all living cells and are vital. They are molecules that carry the information necessary for carrying out activities. Thanks to BDMVs and EOs, intracellular processes are carried out without disrupting cell integrity. It is possible to gain knowledge about nucleic acids, which is a non-invasive procedure. It constitutes an important target for disease detection. In addition, BDMV and It has also been determined in the literature that ESOs can circulate freely in body fluids. 25 This makes sampling easier. On the other hand, the body Isolation of EO and BDMV from fluids and culture medium samples, with the centrifuge-based isolation scheme, which is used as the classical method in the literature When done, it requires advanced and expensive equipment (ultra-fast centrifuge, etc.). This situation makes BDMV and EO-based disease / pathogenicity detection a long process, lasting 30 years. 2 This makes it laborious. Even in this case, the results obtained by classical methods The purity of BDMV and ÖE remains at a low level. Nucleic acid detection used in the characterization of isolated BDMV and OEs. Quantitative Real-Time 5, which is used as the "gold standard" among the leading methods Polymerase Chain Reaction (PC-PCR) is the method used. In this method, nucleic acid... Acid amplification in real time and with the nucleic acid dyes used. This can be tracked using region-specific replicator nucleic acids. The procedure can be performed specifically on the target area. However, this method... due to the high cost of infrastructure and the need for experienced personnel required, 10 It is not possible to implement it in the field and yield results in a short time. Isothermal nucleic acid amplification methods (SNAA) are being developed as an alternative. Unlike methods such as KG-PCR, which require temperature cycles, this is a single These are nucleic acid replication methods that can take place at high temperatures. These characteristics... Thanks to this, INAÇ methods have the potential to be applied in the field and 15 with low device requirements and by any user This can be accomplished. Again, in this method, the necessary steps for analyzing the results are... reaction monitoring using methods that do not require expensive nucleic acid probes. It is possible. Improvements to the classically used BDVM and ÖE isolation methods. and the need for expensive equipment, in addition to the low sample purity, and subsequently the gold standard for detecting nucleic acids in isolated samples The high cost of the KG-PCR method used is a new finding. This shows that the method is needed. 25 United States Law No. US2022098575, which is included in the known state of the art. The patent document describes nucleic acids in ultra-low amounts in biological samples. a system and method that enables the extraction of genetic information from acids It is mentioned. 30 3 Brief Description of the Invention The aim of this invention is to apply isothermal nucleic acid amplification methods to eukaryotic processes. Species identification in exosomes and bacterial outer membrane vesicles, disease and to develop a method that enables the detection of pathogenicity. 5 Detailed Description of the Invention The "Eukaryotic Exosomes and..." project was carried out to achieve the purpose of this discovery. Species Identification, Disease Detection, and Pathogenicity in Bacterial Outer Membrane Vesicles 10 The "Detection Method" is shown in the attached figure; this figure is: Figure 1. Flowchart of the method described in the invention. Figure 2. Microfluidic filtration chip obtained in the method described in the invention. It is a view of the layers. 15 Method 100 Isothermal nucleic acid amplification methods in eukaryotic exosomes and bacterial Species identification in outer membrane vesicles, disease and pathogenicity detection 20 The inventive method that enables its implementation (100); - Preparation of microfluidic filtration chip (101), -for example, species identification will be performed or disease and pathogenicity detection will be carried out. feeding into microfluidic filtration chip (102), -Isothermal nucleic acid test on sample obtained from microfluidic filtration chip 25 The steps of species identification, disease and pathogenicity detection (103) are implemented. It includes. The subject of the invention is the preparation of a microfluidic filtration chip (101) (100) In the next step, preferably 2 mm 30 mm diameter microfluidic chip fabrication will be used. five polymer materials forming layers 1, 3, 5, 8 and 10 of varying thickness 4 (PMMA) layer and polymer layer are the same size and are available in sizes 2, 4, 6, 7 and 9. The layers are made up of 5 layers of double-sided adhesive. DSA (50 μm thick) is cut with a laser cutter. On the 1st and 2nd layers Sample inlet and outlet holes large enough for capillary tube ends to fit into. It is being created. Sample input and output 5 are placed on top of layers 3, 4, 5, 6, 7 and 8. with the holes centered on the inlet and outlet holes. Large holes are being created. Between the holes on layers 8 and 9. A passageway is created connecting the holes. All layers Layers 1 through 10 are stacked on top of each other in sequence, and layers 6 and 7 are... two filters with varying scales of 10-200 nanometers in between They are placed one on top of the other with filters in between. The layers are compressed using a compression mechanism. Layer number 1... Capillary tubes are fixed to the inlet and outlet ports with resin. Microfluidic. The chip consists of a bioreactor chamber and a filter between two plates with holes in it. A bioreactor add-on obtained by installing a magnetic stirrer 15 They are combined. Bacterial cultures are also placed on the sides of the bioreactor attachment. An LED lamp and light sensor are included for use when measuring magnification. The method in question (100) will be used for species determination or disease and pathogenicity (102) 20 In this step, samples are taken directly from patients in liquid form or via microfluidic methods. In the bioreactor section of the chip, the liquid sterile culture medium is combined with a magnetic stirrer. The sample is obtained after homogeneous mixing of the culture medium. The microfluidic chip is connected to the capillary tube at the entry port. Syringe pumps, peristaltic pumps, or 25 that can be used in fluid manipulation using any pump, 1-2 mL volume from the capillary tube at a rate of 40 µL / min. The processed sample is fed into a microfluidic chip. For example, into a microfluidic chip. After the liquid is introduced, filtration takes place during the liquid flow in the chip, and the chip... an isolated vesicle separated from waste materials (debris) at the exit end (isolate or elutant) is obtained, i.e., the separation of biological molecules. 30 is being done. The invention concerns the sample obtained from the (100) microfluidic filtration chip. Species identification, disease and pathogenicity detection through isothermal nucleic acid testing. In step (103), isolated vesicle type obtained from microfluidic chip an isothermal nucleic acid amplification reaction containing specific primers 5 (LAMP, RPA or similar, single-temperature nucleic acid replication) Strain identification is performed by transferring the target reaction. The target used in this stage is... Nucleic acids are found only within the isolated vesicles. For the isolation of biological molecules, cells are precipitated at 10,000 g and 0.2 μm 10 After filtration with a fine-mesh filter, the remaining culture medium supernatant is microfluidic. The chip is passed through the system. In this system, the input part of the microfluidic chip is an example. It is attached to the syringe containing the supernatant, and the outlet end is a 2 mL Eppendorf tube. It is collected within. It will be passed through in a volume of 1 or 2 mL at a rate of 40 μL / minute. For example, the system is then flushed with 1 or 2 mL of PBS. (15 ml) in elutant isothermal nucleic acid assay containing isolated BDMVs It is used. In addition, for the magnification feature on the chip, the microchip itself... A small amount of bacterial cells is injected into the reservoir via cell entry, creating a dynamic effect. by creating a culture environment for them to grow and during this process, they are filtered out through the filtration system. They are enabled to send BDMV. 20 Isothermal reactions model for species-specific detection of isolated BDMVs. The selected LAMP test was performed 10X (according to NEB company protocols). Isothermal Reaction Solution, MgSO4 (100 mM), dNTP Mixture (10 mM), LAMP Primers stock (10X), Bst 2.0 WarmStart DNA Polymerase (NEB, 8 U / 25 WarmStart RTx Reverse Transcriptase (NEB, 0.5) was used for reaction and RNA detection. For the qPCR test, the reaction solution (100 mM / μL) is used. KCl, 160 mM (NH4)2SO4, 20 mM MgSO4, 200 mM Tris-HCl (pH: 8.8), 1% Triton X-100 contains 1 mg / mL Bovine Serum Albumin (10 mm dNTP). The PFU / Psp DNA Polymerase (BIOSWISSTEC) enzyme targets a specific DNA region for 30 seconds. This is done by monitoring the multiplication of the reactions. The analytical monitoring of these two reactions... 6 double-stranded DNA dye (NEB, LAMP Reaction Dye, Thermo Scientific, SYBR Green) or pH-sensitive dyes (Phenol Red) or Magnesium-sensitive The method is to be done with dyes (Hydroxynaphtol Blue) or turbidity test (100) It is performed in step 103. The monitoring time for the relevant LAMP reactions is 45 minutes. For qPCR, the follow-up time depends on the cycle time and the performance of the device (BioRad) 5 Depending on the source, it is estimated to be approximately 2 hours. The meeting topic, centered around these fundamental concepts, is “Eukaryotic Exosomes and Bacterial Species Identification, Disease Detection, and Pathogenicity Detection in External Membrane Vesicles It is possible to develop a wide variety of applications for the "Method (100)", and the invention 10 This cannot be limited to the examples described here, but is primarily stated in the claims. It is like that. 20 30

Claims

7 REQUESTS 1. Isothermal nucleic acid amplification methods and eukaryotic exosomes and Species identification in bacterial outer membrane vesicles, disease and 5 Enables the detection of pathogenicity; - Preparation of microfluidic filtration chip (101), -Species identification will be performed or disease and pathogenicity detection will be carried out. for example, to be given to the microfluidic filtration chip (102), -Isothermal nucleic acid 10 in the sample obtained from the microfluidic filtration chip Species identification, disease and pathogenicity detection are performed through the application of the test. (100) is a method characterized by including steps (103). In step 2, preparation of the microfluidic filtration chip (101), microfluidic Preferably 2 mm thick, 1, 3, 5, 8 and 10. 15 five layers of polymer material (PMMA) that form the layers and 5 that are the same size as the polymer layer and form layers 2, 4, 6, 7 and 9. 1 double-sided adhesive layer (DSA, 50 μm) as in Claim 1, characterized by cutting with a laser cutter (thickness) a method (100). 20 In step 3, microfluidic filtration chip preparation (101), steps 1 and 2. Sample entry point on the layer, large enough for capillary tube tips to enter, and a like the one in Claim 2, characterized by the creation of exit holes method (100). 25 In step (101) of preparing the microfluidic filtration chip, steps 3, 4, 5, 6, 7 and 8. Sample entry and exit holes in the center on the layers holes larger than the inlet and outlet holes to accommodate a method like the one in Claim 2 or 3 characterized by its creation 30 (100). 8 In step 5, microfluidic filtration chip preparation (101), steps 8 and 9. a passage connecting holes between layers Any of Claims 2 to 4 characterized by the creation of a pathway a method like in one (100). 5 In step 6 of preparing the microfluidic filtration chip (101), all layers Stacked in order from 1 to 10, and numbers 6 and 7 two filters with varying scales of 10-200 nanometers between the layers 10 in any of Claims 2 to 5 characterized by placement such a method (100). In step 7, preparation of the microfluidic filtration chip (101), filters are placed between them. a compression of layers stacked on top of each other in such a way that Claims 1 to 6 to 15 are characterized by compression through a mechanism. a method like any of them (100). In step 8, preparation of the microfluidic filtration chip (101), step number 1 by fixing capillary tubes to the entry and exit holes of the layer with resin A method like the one in any of Claims 2 through 7, characterized as 20 (100). In step 9, preparation of microfluidic filtration chip (101), microfluidic between the chip, the bioreactor chamber and two plates with holes in them A bioreactor obtained by installing a filter and magnetic stirrer 25 Any of the 2 to 8 requests characterized by the merging of the extensions a method like in one (100). In step (101) of preparing the microfluidic filtration chip, the bioreactor While measuring the growth of bacterial cultures, also add 30 to the sides of the attachment. 9 characterized by the addition of an LED lamp and a light sensor. A method like any of claims 1 to 9 (100).

11. Species identification will be performed, or disease and pathogenicity detection will be carried out. For example, in step (102) of feeding into the microfluidic filtration chip, 5 samples are taken directly from patients in liquid form or via microfluidic methods. In the bioreactor section of the chip, the liquid is combined with sterile culture medium and magnetically homogeneous mixing of the culture medium with a mixer and then supplying it. as in any of the above characterized claims method (100). 10 12. Species identification will be performed, or disease and pathogenicity detection will be carried out. For example, in step (102) of feeding into the microfluidic filtration chip, for example by bringing the microfluidic chip together with the capillary tube at the entry point A method like the one in Claim 11, characterized (100). 15 13. Species identification will be performed, or disease and pathogenicity detection will be carried out. For example, in step (102) of delivering to the microfluidic filtration chip, syringe pump, peristaltic pump or can be used in fluid manipulation 1-2 mL volume from capillary tube at a rate of 40 µL / min with any pump 20 The process is characterized by the transfer of the processed sample to a microfluidic chip. A method like either 11 or 12 (100).

14. Species identification will be performed, or disease and pathogenicity detection will be carried out. For example, in step (102) of feeding into the microfluidic filtration chip, for example 25 filtering during liquid flow on the chip after microfluidic injection and from the output end of the chip, for example, waste materials (debris) obtaining the separated isolated vesicle, i.e., the biological molecule Any of Claims 11 to 13 characterized by their separation a method like in one (100). 30 15. Isothermal nucleic acid in the sample obtained from the microfluidic filtration chip. Species identification, disease and pathogenicity detection are performed through the application of the test. In step (103), the type of isolated vesicle obtained from the microfluidic chip an isothermal nucleic acid amplification involving specific primers reaction (LAMP, RPA or similar, occurring at a single temperature 5 by transferring (nucleic acid amplification reaction) and determining the strain as in any of the above characterized claims method (100). 15 25

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