- Document Number:
20250011865
- Appl. No:
18/600305
- Application Filed:
March 08, 2024
- نبذة مختصرة :
Provided is a kit for calibration of an isothermal polymerase chain reaction (PCR) analyzer and use thereof. The kit includes a standard substance, an amplification primer set, a reaction buffer, a polymerase, a dye, and a negative control. The standard substance is a DNA plasmid with a gradient concentration of 100 copies/μL to 106 copies/μL; the amplification primer set has nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6; the polymerase is a Bst DNA polymerase; and the dye is a loop-mediated isothermal amplification (LAMP) fluorescent dye (with excitation: 485 nm, emission: 498 nm, and a detection channel of SYBR® Green I or a FAM channel). The kit and a technology for calibration of an isothermal PCR instrument meet calibration demands of the isothermal PCR instrument and fill a technical gap.
- Claim:
1. A primer set for calibration of an isothermal polymerase chain reaction (PCR) analyzer, wherein the primer set comprises the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6.
- Claim:
2. A kit for calibration of an isothermal PCR analyzer, comprising the primer set according to claim 1.
- Claim:
3. The kit according to claim 2, further comprising a standard substance, a reaction buffer, a polymerase, and a dye.
- Claim:
4. The kit according to claim 3, wherein the standard substance is a DNA plasmid with a gradient concentration of 100 copies/μL to 106 copies/μL.
- Claim:
5. The kit according to claim 4, wherein the DNA plasmid is a pMD18 plasmid with a specific sequence shown in SEQ ID NO: 13.
- Claim:
6. The kit according to claim 5, wherein the reaction buffer comprises the following components: 20 mM of Tris-HCl, 10 mM of (NH4)2SO4, 50 mM of KCl, 8 mM of MgSO4, 0.1% of Tween 20, and 1.4 mM each of dNTPs; and the polymerase is a Bst DNA polymerase.
- Claim:
7. The kit according to claim 6, further comprising a negative control.
- Claim:
8. A loop-mediated isothermal amplification (LAMP) reaction system for calibration of an isothermal PCR instrument, wherein the LAMP reaction system has a volume of 25 μL and comprises the following components: 12.5 μL of 2× reaction buffer, 1 μL of Bst DNA polymerase, 0.5 μL of 50×LAMP dye, 5 pmol of SEQ ID NO: 1, 5 pmol of SEQ ID NO: 2, 40 pmol of SEQ ID NO: 3, 40 pmol of SEQ ID NO: 4. 10 pmol of SEQ ID NO: 5, 10 pmol of SEQ ID NO: 6, and 1 μL of the standard substance according to claim 5, and ultrapure water added to make up to 25 μL.
- Claim:
9. A method for calibrating an isothermal PCR analyzer, comprising: designing the primer set according to claim 1 and applying the primer set in the process of calibration of the isothermal PCR analyzer.
- Claim:
10. A method for calibrating an isothermal PCR analyzer, comprising applying the kit according to claim 2 in the process of calibration of the isothermal PCR analyzer.
- Claim:
11. The method according to claim 10, wherein the kit further comprises a standard substance, a reaction buffer, a polymerase, and a dye.
- Claim:
12. The method according to claim 11, wherein the standard substance is a DNA plasmid with a gradient concentration of 100 copies/μL to 106 copies/μL.
- Claim:
13. The method according to claim 12, wherein the DNA plasmid is a pMD18 plasmid with a specific sequence shown in SEQ ID NO: 13.
- Claim:
14. The method according to claim 13, wherein the reaction buffer comprises the following components: 20 mM of Tris-HCl, 10 mM of (NH4)2SO4, 50 mM of KCl, 8 mM of MgSO4, 0.1% of Tween 20, and 1.4 mM each of dNTPs; and the polymerase is a Bst DNA polymerase.
- Claim:
15. The method according to claim 14, wherein the kit further comprises a negative control.
- Current International Class:
12; 12; 12; 12
- الرقم المعرف:
edspap.20250011865
Processing Request
No Comments.