DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of biomedicine, and in specific relates to, a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit that detects latent HIV-1 infections using patient blood samples.
Background of the invention:
[0002] HIV/AIDS is a chronic, potentially life-threatening condition caused by the human immunodeficiency virus (HIV). By damaging your immune system, HIV interferes with your body's ability to fight infection and disease. HIV/AIDS has been a global health issue since the 1980s, claiming human lives each year worldwide. Normally, anti-retroviral therapy (ART) is robust to clear the viral load. However, the viral latency still causes relapses and challenges ART. As of today, there are no medications to treat the latent HIV-1 infections and prevent such relapses.

[0003] RT-PCR (reverse transcription polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) are two different types of laboratory tests used to detect HIV-1 infection. RT-PCR (reverse transcription polymerase chain reaction) is a nucleic acid amplification test that is used to detect the presence of HIV-1 RNA in a sample. RT-PCR is the most sensitive and specific test for HIV infection, and it can detect HIV-1 infection very early after exposure to the virus.

[0004] ELISA (enzyme-linked immunosorbent assay) is an antibody test that is used to detect the presence of HIV-1 antibodies in a sample. HIV-1 antibodies are produced by the body in response to HIV-1 infection. ELISA tests are less sensitive than RT-PCR tests, but they are more widely available and less expensive. There are various methods that are used to detect latent HIV-1 infections includes, polymerase chain reaction (PCR) can be used to detect the presence of HIV-1 DNA in cells and tissues. However, the PCR cannot distinguish between latent and actively replicating HIV-1 infections.

[0005] Viral outgrowth assays (VOAs) are used to measure the size of the latent HIV-1 reservoir. VOAs involve stimulating resting CD4+ T cells to produce new virus. The amount of virus that is produced is a measure of the size of the latent HIV-1 reservoir. Detection of HIV-1 proteins can be detected in cells and tissues using immunohistochemistry or Western blotting. However, detection of HIV-1 proteins cannot distinguish between latent and actively replicating HIV-1 infections.

[0006] The existing techniques and methods cannot detect latent HIV-1 genome integrated into the host genome. Current diagnostic methods for HIV-1 infection primarily rely on the detection of viral RNA or antibodies, which may not reliably identify individuals with latent infections. These methods often require complex laboratory procedures and specialized equipment, limiting their accessibility and widespread adoption. Therefore, there is a need for a simple, sensitive, and cost-effective diagnostic tool for detecting latent HIV-1 infections.

[0007] Therefore, there is a need for a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit that detects latent HIV-1 infections using patient blood samples. There is also a need for a CRlSPR-based diagnostic kit for the detection of latent HIV-1 infections that can precisely detect the presence of the viral genome integrated into the host genome. There is also a need for a CRlSPR-based diagnostic kit for the detection of latent HIV-1 infections that can be used in future as a therapeutic approach to treat patients with latent HIV-1 infections.

[0008] There is also a need for a CRlSPR-based diagnostic kit that enables early identification of individuals with latent infections, facilitating informed treatment decisions, monitoring the effectiveness of HAART and preventing viral rebound and transmission. There is also a need for a CRlSPR-based diagnostic kit that emerges as a promising solution to the limitations of existing latent HIV-1 detection methods. Further, there is also a need for a CRlSPR-based diagnostic kit that improves patient outcomes and reduces the global burden of HIV-1 infection.
Objectives of the invention:
[0009] The primary objective of the invention is to provide a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit that detects latent HIV-1 infections using patient blood samples.

[0010] Another objective of the invention is to provide a CRlSPR-based diagnostic kit that accurately detects latent HIV-1 genome integrated into the host genome.

[0011] The other objective of the invention is to provide a CRlSPR-based diagnostic kit that identifies latent viral reservoirs and viral genome-containing host cells.

[0012] Yet another objective of the invention is to provide for a CRlSPR-based diagnostic kit that enables in early identification of individuals with latent infections, facilitating informed treatment decisions, monitoring the effectiveness of HAART and preventing viral rebound and transmission.

[0013] Further objective of the present invention is to provide a CRlSPR-based diagnostic kit for the detection of latent HIV-1 infections that can be used in future as a therapeutic approach to treat patients with latent HIV-1 infections.
Summary of the invention:
[0014] The present disclosure proposes a diagnostic kit for the detection of latent HIV-1 infections using patient blood samples. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0015] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit that detects latent HIV-1 infections using patient blood samples.

[0016] According to an aspect, the invention provides a CRlSPR-based diagnostic kit for the detection of latent HIV-1 infections using patient blood samples. In one embodiment herein, a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit (CHIKit-SA) for detecting latent HIV-1 infection in patients comprises a genomic DNA isolation kit, a CRISPR-Cas9 system and an agarose gel electrophoresis unit.

[0017] In one embodiment herein, the genomic DNA isolation kit is configured for isolating genomic deoxyribonucleic acid (DNA) from a blood sample of a patient. The genomic DNA isolation kit utilizes buffers, a centrifuge and an incubator. In one embodiment herein, the CRISPR-Cas9 system is configured to detect and cleave specific HIV-1 gene integrated in isolated DNA. The CRISPR-Cas9 system comprises a guide ribonucleic acid and a Cas9 enzyme.

[0018] In one embodiment herein, the guide ribonucleic acid (gRNA) is configured to identify the specific sequence of HIV-1 gene within the isolated DNA. In one embodiment herein, the Cas9 enzyme is configured to cleave the HIV-1 gene at identified location. In one embodiment herein, the agarose gel electrophoresis unit is configured to separate and visualize DNA fragments to confirm presence of cleaved HIV-1 DNA. The agarose gel electrophoresis unit includes an agarose gel, an electrophoresis buffer, an electrophoretic system, a power pack, the isolated genomic DNA and a DNA ladder.

[0019] According to another aspect, the invention provides a method for detecting latent HIV-1 infection in the patient. At one step, the blood sample of at least 1 ml is collected from the patient using a syringe and storing the blood sample using a tube. At one step, a genomic deoxyribonucleic acid (DNA) is isolated from the blood sample using a genomic DNA isolation kit. At one step, the isolated genomic DNA is combined with the CRISPR-Cas9 system and buffered in a vial for preparing a mixture, thereby incubating the mixture at a predetermined temperature. In addition, the CRISPR-Cas9 system includes Cas9 enzyme and, guide ribonucleic acid (gRNA).

[0020] At one step, the incubated mixture is analyzed for a time period ranging between 45 - 60 minutes using the agarose gel electrophoresis unit and the analysed gel is visualized in a UV transilluminator to identify Cas9 enzyme activity on the target HIV-1 sequence. At one step, positive results of the analysed gel are sequenced in the agarose gel electrophoresis unit to identify specific virus strains, thereby confirming the presence of latent HIV-1 infection. In one embodiment herein, the genomic DNA isolation kit performs quality check analysis for the isolated genomic DNA using the agarose gel electrophoresis unit.

[0021] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0022] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0023] FIG. 1 illustrates a block diagram of a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit for the detection of latent HIV-1 infections using patient blood samples, in accordance to an exemplary embodiment of the invention.

[0024] FIG. 2 illustrates a flowchart of a method for detecting latent HIV-1 infection in the patient, in accordance to an exemplary embodiment of the invention.

[0025] FIG. 3 illustrates a pictorial representation of genomic DNA samples of patient-1 and patient-2 obtained in an UV transilluminator, in accordance to an exemplary embodiment of the invention.

[0026] FIG. 4 illustrates a pictorial representation of an agarose gel with HIV patient gene amplified, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0027] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0028] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit that detects latent HIV-1 infections using patient blood samples.

[0029] According to an exemplary embodiment of the invention, FIG. 1 illustrates a block diagram of a clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic kit 100 for the detection of latent HIV-1 infections using patient blood samples. In one embodiment herein, a clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit (CHIKit-SA) 100 for detecting latent HIV-1 infection in patients comprises a genomic DNA isolation kit 102, a CRISPR-Cas9 system 104 and an agarose gel electrophoresis unit 106.

[0030] In one embodiment herein, the genomic DNA isolation kit 102 is configured for isolating genomic deoxyribonucleic acid (DNA) from a blood sample of a patient. The genomic DNA isolation kit 102 is a specialized set of reagents and protocols designed to extract high-quality genomic DNA from various biological samples, such as blood, tissue, or cells. The isolation of genomic DNA is a crucial step in many molecular biology and genetic analysis techniques, including PCR, sequencing, and genotyping. The genomic DNA isolation kit 102 utilizes buffers, a centrifuge and an incubator.

[0031] In one embodiment herein, the buffers assist in disrupting cellular membranes, proteins, and other cellular components. The buffers include detergents, salts, and other chemicals tailored to the blood sample. Later a lysis solution is added to the blood sample to break down cell membranes and release genomic DNA. Next, a proteinase K is added to the genomic DNA to obtain a lysate by degrading proteins.

[0032] Next, the lysate is transferred to a spin column for performing a centrifuge action to bind while contaminants are washed away. Later, the spin column is washed with wash buffers to remove residual contaminants. Next, the purified genomic DNA is eluted from the column or beads using an elution buffer. Finally, the concentration of the purified genomic DNA is measured and the quality of the purified genomic DNA is assessed using spectrophotometry or other methods.

[0033] In one embodiment herein, the CRISPR-Cas9 system 104 is configured to detect and cleave a specific HIV-1 gene integrated in isolated DNA. The CRISPR-Cas9 system 104 comprises a guide ribonucleic acid and a Cas9 enzyme. In one embodiment herein, the guide ribonucleic acid (gRNA) is configured to identify the specific sequence of the HIV-1 gene within the isolated DNA. In one embodiment herein, the Cas9 enzyme is configured to cleave the HIV-1 gene at an identified location. The Cas9 enzyme plays a major role in the CRISPR-Cas9 system’s ability to target and modify the specific DNA sequence.

[0034] In one embodiment herein, the guide ribonucleic acid (gRNA) comprises a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In one embodiment herein, the CRISPR RNA (crRNA) is a short RNA sequences that are complementary to target DNA sequence. Each crRNA contains a unique guide sequence that directs the Cas9 enzyme to the specific region of the DNA to be modified. The tracrRNA is another RNA molecule that forms a complex with the crRNA and assists in activating the Cas9 enzyme. The Cas9 enzyme is an RNA-guided endonuclease enzyme that is responsible for cutting the DNA at the target location specified by the crRNA. It forms a complex with the crRNA and tracrRNA to form the CRISPR-Cas9 ribonucleoprotein (RNP) complex.

[0035] In one embodiment herein, the agarose gel electrophoresis unit 106 is configured to separate and visualize DNA fragments to confirm presence of cleaved HIV-1 DNA. The agarose gel electrophoresis unit 106 includes an agarose gel, an electrophoresis buffer, an electrophoretic system, a power pack, the isolated genomic DNA and a DNA ladder.

[0036] In one embodiment herein, the agarose gel is prepared by dissolving an agarose powder in the electrophoresis buffer to obtain a form a solution. The electrophoresis buffer includes tris-acetate-EDTA (TAE) and tris-borate-EDTA (TBE). Next, the solution is heated until the agarose is completely dissolved. Later, pour the heated solution into a gel tray and insert a comb to create wells for collected DNA blood samples. Next, the DNA blood samples are mixed with loading buffer, thereby loading them into the wells using a micropipette to ensure equal volumes of the DNA blood samples are loaded into each well.

[0037] The gel tray with electrodes at each end holds the solution in place during electrophoresis. The electrodes are prepared by platinum and graphite, which are connected to the power pack and generate the necessary electrical power for mitigating DNA. The power pack enables a user to control the voltage and current applied to the solution, thereby influencing the speed and resolution of DNA migration.

[0038] In one embodiment herein, the gel tray is submerged in the electrophoresis buffer and connected to the power pack. A voltage is applied across the solution and run the electrophoresis for a predetermined period, typically at a constant voltage. After electrophoresis, remove the solution from the agarose gel electrophoresis unit 106 and strain the DNA blood sample with a fluorescent dye such as ethidium bromide or SYBR safe to monitor progress of electrophoresis, thereby assisting to visualize DNA migration. In addition, the DNA bands are visualized using the UV transilluminator.

[0039] In one embodiment herein, the DNA ladder is also known as DNA size marker and molecular weight marker, which consists of a mixture of DNA fragments of known sizes. It is loaded onto the solution alongside the DNA blood samples to estimate the size of DNA fragments in the samples.

[0040] According to another embodiment of the invention, FIG. 2 refers to a flowchart 200 of a method for detecting latent HIV-1 infection in the patient. At step 202, the blood sample of at least 1 ml is collected from the patient using a syringe and the blood sample is stored using a tube. At step 204, a genomic deoxyribonucleic acid (DNA) is isolated from the blood sample using the genomic DNA isolation kit 102. At step 206, the isolated genomic DNA is combined with the CRISPR-Cas9 system 104 and buffered in a vial for preparing a mixture, thereby incubating the mixture at a predetermined temperature. In addition, the CRISPR-Cas9 system 104 includes Cas9 enzyme and, guide ribonucleic acid (gRNA).

[0041] At step 208, the incubated mixture is analyzed for a time period ranging between 45 - 60 minutes using the agarose gel electrophoresis unit 106 and the analysed gel is visualized in a UV transilluminator to identify Cas9 enzyme activity on the target HIV-1 sequence. At step 210, positive results of the analysed gel are sequenced in the agarose gel electrophoresis unit 106 to identify specific virus strains, thereby confirming the presence of latent HIV-1 infection. The genomic DNA isolation kit 102 performs quality check analysis forthe isolated genomic DNA using the agarose gel electrophoresis unit 106.

[0042] According to another embodiment of the invention, FIG. 3 refers to a pictorial representation 300 of genomic DNA samples of patient-1 and patient-2 obtained in an UV transilluminator. In one embodiment herein, the patient-1 genomic DNA samples are isolated into three separate vials, likely represented by three lanes in the top row. In one embodiment herein, the patient-2 genomic DNA samples are isolated into five separate vials, likely represented by the five lanes in the bottom row.

[0043] The first lane in both the top and bottom rows of the genomic DNA sample contains a DNA ladder. In addition, the DNA ladder is a mixture of DNA fragment of known sizes, used as a reference to estimate the size of DNA fragments in the DNA samples. In one embodiment herein, the pattern of the DNA blood sample in each lane can reveal information about the size distribution of DNA fragments in the DNA blood samples.

[0044] According to another embodiment of the invention, FIG. 4 refers to a pictorial representation 400 of an agarose gel with HIV patient gene amplified. In one embodiment herein, the first lane contains a 1 kb DNA and the second lane contains a 100 bp DNA ladder. These ladders serve as reference for estimating the size of the DNA fragments in the blood samples. Here, third lane shows the blood sample of a healthy individual, which serves as a negative control. In this lane, no band corresponding to the HIV gene is visible, thereby indicating the absence of the HIV gene in the healthy person's genomic DNA blood sample.

[0045] In one embodiment herein, fourth and fifth lanes contains blood samples from the two HIV patients, as depicted as P1 and P2, respectively, as shown in FIG. 4. Thick and bright bands are observed in these lanes, indicating the presence of the HIV gene in the genomic DNA samples obtained from the HIV patients. Here, the DNA ladders in the first and second lanes serve as size markers, thereby allowing for estimating sizes of DNA fragments observed in the blood samples collected from patients.

[0046] The absence of a band corresponding to the HIV gene in the healthy person sample (third lane) indicates a negative result, thereby confirming that the individual does not carry the HIV gene. The presence of thick and bright bands in the fourth and fifth lanes corresponds to the DNA blood samples from HIV patients P1 and P2, respectively, thereby indicates a positive result for the presence of the HIV gene in these patients' genomic DNA samples.

[0047] The intensity and size of the bands may vary between samples, but the presence of any visible band suggests the presence of the HIV gene in the patient's genomic DNA. The agarose gel analysis confirms the presence of the HIV gene in the genomic DNA samples obtained from patients P1 and P2, while the sample from the healthy individual shows no evidence of the HIV gene.

[0048] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a diagnostic kit for the detection of latent HIV-1 infections using patient blood samples is disclosed. The proposed clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit 100 detects latent HIV-1 infections using patient blood samples. The proposed CRlSPR-based diagnostic kit 100accurately detects latent HIV-1 genome integrated into the host genome.

[0049] The proposed CRlSPR-based diagnostic kit 100 identifies latent viral reservoirs and viral genome-containing host cells. The proposed CRlSPR-based diagnostic kit 100 enables in early identification of individuals with latent infections, facilitating informed treatment decisions, monitoring the effectiveness of HAART and preventing viral rebound and transmission. The proposed CRlSPR-based diagnostic kit 100 is utilized for the detection of latent HIV-1 infections that can be used in future as a therapeutic approach to treat patients with latent HIV-1 infections.

[0050] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
,CLAIMS:CLAIMS:
I / We Claim:
1. A clustered regularly interspaced short palindromic repeats (CRlSPR)-based diagnostic kit (CHIKit-SA) (100) for detecting latent HIV-1 infection in patients, comprising:
a genomic DNA isolation kit (102) configured for isolating genomic deoxyribonucleic acid (DNA) from a blood sample of a patient;
a CRISPR-Cas9 system (104) configured to detect and cleave specific HIV-1 gene integrated in isolated DNA, wherein the CRISPR-Cas9 system (104) comprises:
a guide ribonucleic acid (gRNA) configured to identify the specific sequence of HIV-1 gene within the isolated DNA; and
a Cas9 enzyme configured to cleave the HIV-1 gene at identified location;
an agarose gel electrophoresis unit (106) configured to separate and visualize DNA fragments to confirm presence of cleaved HIV-1 DNA.
2. The CRISPR-based diagnostic kit (CHIKit-SA) (100) as claimed in claim 1, wherein the genomic DNA isolation kit (102) utilizes buffers, a centrifuge and an incubator.
3. The CRISPR-based diagnostic kit (CHIKit-SA) (100) as claimed in claim 1, wherein the agarose gel electrophoresis unit (106) includes an agarose gel, an electrophoresis buffer, an electrophoretic system, a power pack, the isolated genomic DNA and a DNA ladder.
4. A method for detecting latent HIV-1 infection in a patient, comprising:
collecting a blood sample from the patient using a syringe and storing the blood sample using a tube;
isolating a genomic deoxyribonucleic acid (DNA) from the blood sample using a genomic DNA isolation kit (102);
combining isolated genomic DNA with a CRISPR-Cas9 system (104) and buffers in a vial for preparing a mixture, thereby incubating the mixture at a predetermined temperature;
analyzing incubated mixture using an agarose gel electrophoresis unit (106) and visualizing the analysed gel in a UV transilluminator to identify Cas9 enzyme activity on the target HIV-1 sequence; and
sequencing positive results of the analysed gel in the agarose gel electrophoresis unit (106) to identify specific virus strains, thereby confirming the presence of latent HIV-1 infection.
5. The method as claimed in claim 4, wherein the CRISPR-Cas9 system (104) includes Cas9 enzyme and, guide ribonucleic acid (gRNA).
6. The method as claimed in claim 4, wherein the blood sample of at least 1 ml is collected from the patient.
7. The method as claimed in claim 4, wherein the genomic DNA isolation kit (102) performs quality check analysis for the isolated genomic DNA using the agarose gel electrophoresis unit (106).
8. The method as claimed in claim 4, wherein the incubated mixture is analysed using the agarose gel electrophoresis unit (106) at a time period varies between 45 – 60 minutes.