TH17821U - Primers for detecting the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus using real-time PCR. - Google Patents

Abstract

------14/06/2018------(OCR) Page 1 of 1. Abstract of the Invention: This invention is a primer for detecting the immunodominant staphylococcal antigen A (isoA) gene in methicillin-resistant Staphylococcus oureus using real-time PCR. It consists of two primers capable of specifically binding to the isoA gene sequence on the target DNA. The primers are mixed with a test reagent and combined with a sample of genetic material, complementary DNA (cDNA), or RNA isolated from the bacteria sample. The genetic material is then amplified using real-time PCR. This invention can be used for both qualitative and quantitative analysis. For the study of gene expression that provides accurate, reliable, highly sensitive and specific results, and can report results quickly ------------ Page 1 of 1 page Summary of the Invention This invention is a primer and reagent kit for the detection of the immunodominant staphylococcal antigen A (1isoA) gene in methicillin-resistant Staphylococcus aureus using real-time PCR. It consists of two primers that can specifically bind the base sequence of the isaA gene on the target DNA. The reagent kit is for testing with the primers by mixing with a sample of genetic material or complementary DNA (cDNA) from RNA isolated from the bacterial sample. The genetic material was then amplified using real-time PCR. This invention can be used for both qualitative and quantitative analysis, particularly in gene expression studies, providing accurate, reliable, highly sensitive, and specific results that can be reported quickly.

Claims (4)

Revised 26/10/2018. Claim 1. Primers for the detection of the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus using real-time PCR. This consists of a pair of primers used to amplified the genetic material for the immunodominant gene. Two primers for detecting the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus. Forward primer: 5'-GCA GTA TTA CCA TTG CTT A-3'. Reverse primer: 5'-GCA CAA TCA AGT AAC TCA-3'. ------- ------14/06/2018------(OCR) Page 1 of 2. Claim 1. Primers for detecting the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus. (methicillin-resistant Staphylococcus aureus) using real-time PCR technique, which consists of a pair of primers used to amplified the genetic material for the immunodominant staphylococcal antigen A (isaA) gene, 2 strands with the following base sequences: forward primer: 5’-GCA GTA TTA CCA TTG CTT A-3’, reverse primer: 5’-GCA CAA TCA AGT AAC TCA-3’. 2. Primers for detecting the immunodominant staphylococcal antigen A (isaA) gene for methicillin-resistant Staphylococcus aureus. (methicillin-resistant Staphylococcus aureus) using real-time PCR technique according to Patent 1, where the optimal primer and reagent quantities for testing and reaction are the primer pairs for the immunodominant gene. The following quantities of primers are needed: 1.0 µl each of 4 micromolar primers for the isaA gene; 1.0 µl each of 4 micromolar primers for the 16S rRNA gene; 20.0 µl of PCR reaction mix (for reacting with the isaA primers and 10.0 µl each of the 16S rRNA primers); and 12.0 µl of deionized distilled water (for reacting with the isaA primers and 6.0 µl each of the 16S rRNA primers). 3. Primers for detecting the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus using real-time PCR technique according to Patent 1 or 2, where the amplification of the genetic material and verification of the PCR product using a real-time PCR machine have the following temperature programming conditions: Step 1: Pre-denature program using 95°C for 10 minutes (Page 2 of 2). Step 2: Amplification program. (Amplification) consists of 3 steps: denaturation at 95°C for 20 seconds, annealing at 53°C for 20 seconds, and extension at 72°C for 30 seconds. The fluorescence signal is measured once. This process is repeated 45 times, with a temperature transition rate of 20°C per second. Step 3, the melting curve program, consists of 3 steps: increasing the temperature to 95°C for 15 seconds, then decreasing it to 60°C for 60 seconds using a temperature transition rate of 20°C per second, and then increasing the temperature back to 95°C for 15 seconds using a temperature transition rate of 0.1°C per second, and measuring the fluorescence signal. Continuously, Step 4: Cooling program reduces the temperature to 40 degrees Celsius for 30 seconds using a temperature change rate of 20 degrees Celsius per second. ------------ Page 1 of 2 pages. Claims 1. Primers and reagents for detecting the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus using real-time PCR. These consist of two primers used to amplified the genetic material for the immunodominant staphylococcal antigen A (isaA) gene, with the following base sequences: Forward primer: 5’-GCA GTA TTA CCA TTG CTT A-3’; Reverse primer: 5’-GCA CAA TCA AGT AAC TCA-3’. 2. Primers and reagent kits for the detection of the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus using real-time PCR, as per claim 1, which may be supplemented with a reagent kit for the detection of the immunodominant staphylococcal antigen A (isaA) gene in methicillin-resistant Staphylococcus aureus. (methicillin-resistant Staphylococcus aureus) which consists of: a. Two primers for the 16S rRNA gene, which is used for normalization, with the following base sequences: Forward primer: 5’-GCC TAA TAC ATG CAA GTC-3’; Reverse primer: 5’-CCA GTC TTA TAG GTA GGT TA-3’. b. PCR reaction mix, which is a commercial brand name: Power SYBR® Green PCR Master Mix. c. Deionized distilled water of PCR grade purity, used to adjust the test reagent volume to the final volume. 3. Primers and reagents for the detection of the immunodominant staphylococcal antigen A (isaA) gene for methicillin-resistant Staphylococcus aureus using real-time PCR technique according to claim 1 or 2, where the optimal amount of primers and reagents for testing and reaction is shown on page 2 of 2 pages. Primer pairs for the immunodominant gene. Immunodominant staphylococcal antigen A (isoA) gene, with 4 micromolar concentration at each primer, 1.0 µl each. Primers for the 16S rRNA gene, with 4 micromolar concentration at each primer, 1.0 µl each. PCR reaction mix, 20.0 µl (for reacting with the isoA primers and 10.0 µl each of the 16S rRNA primers). Deionized distilled water, 12.0 µl (for...) (React with 6.0 μL each of primers for the isoA gene and the 16S rRNA gene.) 4. Primers and reagents for the detection of the immunodominant staphylococcal antigen A (isoA) gene for methicillin-resistant Staphylococcus aureus using real-time PCR technique according to one of the three claims 1-3, where the amplification of the genetic material and verification of the PCR product using a real-time PCR machine have the following temperature programming conditions: Step 1: Pre-denature program at 95°C for 10 minutes. Step 2: Amplification program... (Amplification) consists of 3 steps: denaturation at 95°C for 20 seconds, annealing at 53°C for 20 seconds, and extension at 72°C for 30 seconds. The fluorescence signal is measured once. This process is repeated 45 times, with a temperature transition rate of 20°C per second. Step 3, the melting curve program, consists of 3 steps: increasing the temperature to 95°C for 15 seconds, then decreasing it to 60°C for 60 seconds using a temperature transition rate of 20°C per second, and then increasing the temperature back to 95°C for 15 seconds using a temperature transition rate of 0.1°C per second. The fluorescence signal is then measured. Continuously, step 4, the cooling program, reduces the temperature to 40 degrees Celsius for 30 seconds using a temperature change rate of 20 degrees Celsius per second.