TH14896U - Primers for detecting the fungus Malassezia globosa. - Google Patents

Abstract

------July 26, 2018------(OCR) Page 1 of 1 page. Summary of the Invention. The primer kit for detecting Malassezia globosa is a set of two primers used to detect species-level DNA of Malassezia globosa using the polymerase chain reaction (PCR) technique. The primer sequences are as follows: - Forward primer: 5’- cgt aca ata agt gtg tct ctg gcg -3’ - Reverse primer: 5’- gtt teg ctg cgt tct tea teg -3’ Additional reagents for PCR testing of Malassezia globosa can also be added.

Claims (4)

------July 26, 2018------(OCR) Page 1 of 1 page. Claims. 1. Primers for detecting Malassezia globosa fungus consist of pairs of primers used to amplify the genetic material (DNA) of Malassezia globosa. The base sequences are as follows: Forward primer: 5’- cgt aca ata agt gtg tct ctg gcg -3’; Reverse primer: 5’- gtt tcg ctg cgt tct tca tcg -3’. 2. A method for amplifying the genetic material of the fungus Malassezia globosa using primers for detecting Malassezia globosa as per claim 1, using the polymerase chain reaction (PCR) technique with a thermal cycler (PCR machine). The temperature program for DNA amplification is as follows: Step 1: Pre-denature program at 94 degrees Celsius for 3 minutes. Step 2: Amplification program... (Amplification) Consists of 3 steps: Denaturation at 94°C for 45 seconds; Annealing at 62°C for 30 seconds; and Extension at 72°C for 30 seconds. This is repeated 30 cycles, with a temperature transition rate of 5°C per second on the thermal cycler. Step 3: Final Extension program, using 72°C for 4 minutes. Step 4: Cooling program, reducing the temperature to 4°C (unlimited time). ------28/12/2017------(OCR) Page 1 of 2. Claims. 1. Primers for detecting the fungus Malassezia globosa consist of pairs of primers used to amplify the genetic material (DNA) of the fungus Malassezia globosa. The base sequences are as follows: Forward primer: 5’- cgt aca ata agt gtg tct ctg gcg -3’; Reverse primer: 5’- gtt teg ctg cgt tct tea teg -3’. 2. A reagent kit for detecting the fungus Malassezia globosa, which is used in conjunction with... Together with the primers as per claim 1, the kit for the detection of the fungus Malassezia globosa contains 10XPCR buffer containing 50 millimolar potassium chloride (mM KCl), 10 millimolar tris-hydrochloride (mM Tris-HCI pH 8.3), and 1-1.5 millimolar magnesium chloride (mM MgCl2); a dNTPs mix containing four dNTPs: dATP, dCTP, dGTP, and dTTP at a concentration of 10 millimolar; and a magnesium chloride reagent. (MgCl2) 50 millimolar concentration, Taq DNA polymerase 5 units per microliter, deionized distilled water of PCR grade purity. 3. Primers and reagent kits for the detection of Malassezia globosa fungus according to claim 1 or 2, where the primer pair used to amplify the DNA of Malassezia globosa fungus contains the most suitable master mix for one reaction, namely: a primer pair for amplifying the DNA of Malassezia globosa fungus consisting of a forward primer and a reverse primer with a concentration of 10 micromolar, each with a volume of 0.6 microliters; a PCR reaction mix consisting of a 10X concentration PCR buffer with a volume of 3 microliters; and all four types of dNTPs, namely dATP, 0.6 µL of 10 mmol/dCTP, dGTP, and dTTP solution, 0.6 µL of 50 mmol/dGCL solution, and 0.2 µL of Taq DNA polymerase enzyme at a concentration of 5 units per µL were used. Genetic material extracted from samples at concentrations of 1-50 nanograms was adjusted with deionized distilled water (PCR grade) to a final volume of 30 µL. 4. Primers and reagent kits for the detection of Malassezia globosa fungus according to any one of the claims 1-3, where the amplification of genetic material (DNA) by polymerase chain reaction (PCR) using a thermal cycler (PCR machine) has a program temperature condition for DNA amplification according to the following steps: Step 1: Pre-denature program at 94 degrees Celsius for 3 minutes. Step 2: Amplification program. (Amplification) Consists of 3 steps: Denaturation at 94°C for 45 seconds; Annealing at 62°C for 30 seconds; and Extension at 72°C for 30 seconds. This is repeated 30 cycles, with the thermal cycler's temperature transition rate being 5°C per second. Step 3: Final Extension program, using 72°C for 4 minutes. Step 4: Cooling program, reducing the temperature to 4°C (unlimited time). 1. The primer and reagent kit for detecting Malassezia globosa consists of primer pairs used to amplify the genetic material (DNA) of Malassezia globosa. The primers have the following sequences: Forward primer: 5' - cgt aca ata agt gtg tct ctg gcg -3'; Reverse primer: 5' - gtt tcg ctg cgt tct tca tcg -3'. 2. Primers and reagent kits for the detection of Malassezia globosa, as per claim 1, which may be supplemented with reagent kits for the diagnostic reaction of Malassezia globosa, consisting of: The 10XPCR buffer solution contains 50 millimolar potassium chloride (mM KCl), 10 millimolar tris-hydrochloride (mM Tris-HCI pH 8.3), and 1-1.5 millimolar magnesium chloride (mM MgCl2). The dNTPs mix solution contains four dNTPs: dATP, dCTP, dGTP, and dTTP at a concentration of 10 millimolar. A magnesium chloride (MgCl2) solution is also included at a concentration of 50 millimolar. The Taq DNA polymerase solution is also present. A concentration of 5 units per microliter of deionized distilled water with a PCR purity level. 3. Primers and reagent kits for the detection of Malassezia globosa fungus according to claim 1 or 2, where the primer pair used to amplify the DNA of Malassezia globosa fungus contains the most suitable amount of master mix for one reaction, namely: a primer pair for amplifying the DNA of Malassezia globosa fungus consisting of a forward primer and a reverse primer with a concentration of 10 micromolar, each with a volume of 0.6 microliters; a PCR reaction mix consisting of a 10X concentration PCR buffer with a volume of 3 microliters; and all four types of dNTPs, namely... dATP, dCTP, dGTP, and dTTP at a concentration of 10 millimolar, with a volume of 0.6 microliters; magnesium chloride (MgCl2) solution at a concentration of 50 millimolar, with a volume of 0.6 microliters; and the enzyme Taq DNA polymerase at a concentration of 5 units per microliter, with a volume of 0.2 microliters, were used. Genetic material extracted from samples at concentrations of 1-50 nanograms was adjusted with deionized distilled water of PCR grad to a final volume of 30 microliters. 4. Primers and reagent kits for the detection of Malassezia globosa fungus according to any one of the claims 1-3, where the amplification of genetic material (DNA) by the polymerase chain reaction (PCR) technique using a thermal cycler (PCR machine) has the following temperature program conditions for DNA amplification: Step 1: Pre-denature program at 94 degrees Celsius for 3 minutes; Step 2: Amplification program. (Amplification) Consists of 3 steps: Denaturation at 94°C for 45 seconds; Annealing at 62°C for 30 seconds; and Extension at 72°C for 30 seconds. This is repeated 30 cycles, with the thermal cycler's temperature transition rate being 5°C per second. Step 3: Final Extension program, using 72°C for 4 minutes. Step 4: Cooling program, reducing the temperature to 4°C (no time limit).