Primer set and procedure for preparing hepatitis C virus (N3) and NS5A genes using single-step multiplex nested polymerase chain reaction (SPR) method.

Abstract

OCR 10KL (21 / 08 / 2568) Primer set for the single-step preparation of NS3 and NS5A genes of hepatitis C virus. Multiplex nested polymerase chain reaction (Single-step Multiplex nested Polymerase Chain) The reaction consists of primers used for preparing the NS3 and NS5A genes. Eight hepatitis C virus variants were collected and their genetic material was amplified using the single-step method. Multiplex nested polymerase chain reaction (Single-step Multiplex nested Polymerase Chain) (Reaction) which can amplify multiple target genes with multiple primer pairs simultaneously in the same in-tube reaction. The ability to prepare both NS3 and NS5A genes of the hepatitis C virus makes this a good, convenient, rapid, and suitable method. than

Claims

OCR 10KL (21 / 08 / 2568) 1. Primer set for preparing NS3 and NS5A genes of hepatitis C virus using the single-step multiplex method. Nested Polymerase Chain Reaction (Single-step Multiplex nested Polymerase Chain Reaction) This consists of primers used for preparing the NS3 and NS5A genes of the hepatitis C virus. Eight strands with the following base sequence: The forward primer for the outer region of the NS3 gene is 5'-attaccgggagatgggttg-3'. The reverse primer for the outer region of the NS3 gene is 5'-ttgaattgtcagagaaggatgg-3'. The forward primer for the outer region of the NS5A gene is 5'-actggctgcgtaccatctg-3'. The reverse primer for the outer region of the NS5A gene is 5'-caacatcgaggtcagcacag-3'. The forward primer for the NS3 gene (inner portion) is 5'-gcttgactggcagggataag-3'. The reverse primer for the NS3 gene's inner portion is 5'-acacagcagcccgaaagat-3'. The forward primer for the NS5A gene in the inner portion is 5'-ggtttgcatggtgttgtcts-3'. The reverse primer for the NS5A gene is 5'-cctattgcgtaggagttcaacc-3'.

2. Procedure for preparing NS3 and NS5A genes of hepatitis C virus using the single-step multiplex method. Nested Polymerase Chain Reaction (Single-step Multiplex nested Polymerase Chain Reaction) Using the primer kit as per claim 1, the following steps are involved: a. Extract viral nucleic acid (RNA) from serum samples. B. Synthesize cDNA from hepatitis C virus RNA samples using the reverse transcription method. (reverse transcription) C. Prepare the NS3 and NS5A genes of hepatitis C virus using the single-step multiplex nested method. Single-step Multiplex nested Polymerase Chain Reaction Using primers for the NS3 and NS5A outer genes, and under optimal conditions for... Prepare the NS3 and NS5A outer regions of the genes by performing first-round PCR, which means preparing... A PCR reaction mix consisting of 1x PCR buffer. (PCR buffer), 100-500 micromolar. The four types of dNTPs: dATP, dCTP, dGTP and dTTP and the enzyme Taq DNA polymerase. At a concentration of 0.5-2.0 units (U), add the primer for preparing the NS3 and NS5A outer layer. With each primer having a final concentration of 0.25-1.5 micromolar, add the DNA sample. Add 0.5-10 microliters of water and then add pure distilled water, adjusting until the final volume is equal to... 25 microliters were then fed into the PCR (Thermal cycle) machine, with the temperature programmed as follows: Step 1: Pre-incubation program using a temperature of 94 degrees Celsius for 7 minutes. Step 2: Amplification program, which consists of 3 steps: Denaturation is performed at 94 degrees Celsius for 10 seconds. The annealing process uses a temperature of 45 degrees Celsius for 30 seconds. The extension step uses a temperature of 68 degrees Celsius for 40 seconds. Repeat this for a total of 40 cycles. Step 3: The Final Extension program uses a temperature of 68 degrees Celsius. 7 minutes d. Prepare the NS3 and NS5A genes of hepatitis C virus using the single-step multiplex nested method. Single-step Multiplex nested Polymerase Chain Reaction Using primers for the NS3 and NS5A genes and under optimal conditions, The NS3 and NS5A genes were prepared using second-round PCR, which involves preparing... A PCR reaction mix consisting of 1x PCR buffer. buffer), 100-500 micromolar. The four types of dNTPs are dATP, dCTP, dGTP. And dTTP and the enzyme Taq DNA polymerase. A concentration of 0.5–2.0 units (U) was used to add primers for the preparation of NS3 and NS5A genes. Each primer was given a final concentration of 0.25-1.5 micromolar, and the PCR product was added. (PCR product) 0.2-5.5 microliters, and add purified distilled water to the desired volume. Finally, the amount equals 25 microliters, which is then fed into a PCR (thermal cycle) machine according to the program. The temperature is as follows: Step 1: Pre-incubation program using a temperature of 94 degrees Celsius for 7 minutes. minute Step 2: Amplification program, which consists of 3 steps: Denaturation is performed at 94 degrees Celsius for 30 seconds. The annealing process uses a temperature of 46 degrees Celsius for 30 seconds. The extension step uses a temperature of 72 degrees Celsius for 40 seconds. Repeat this for a total of 40 cycles. Step 3: The Final Extension program uses a temperature of 72 degrees Celsius. 7 minutes Step 4: The obtained PCR product is then subjected to DNA size separation using a 1.0-1.5% agarose solution. Gel electrophoresis yields a DNA band of 417 base pairs representing the NS3 gene and another DNA band containing... The size is 522 base pairs for the NS5A gene.

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