Methods and systems for PCR quantitation

Patent metadata

Field	Value
Publication number	US-10385386-B2
Application number	US-201515301947-A
Country	US
Kind code	B2
Filing date	Apr 3, 2015
Priority date	Apr 4, 2014
Publication date	Aug 20, 2019
Grant date	Aug 20, 2019

Abstract

A method for quantifying nucleic acid is provided. The method includes determining a first reference threshold cycle for a first predetermined input quantity for a reference nucleic acid, determining a first target threshold cycle for the first predetermined input quantity for a target nucleic acid, determining a second reference threshold cycle for a second predetermined input quantity for the reference nucleic acid, and determining a second target threshold cycle, by the processor, for the second predetermined input quantity for the target nucleic acid. The method further includes receiving a sample threshold cycle, determining a sample input quantity based on the first and second reference threshold cycle and the first and second target threshold cycle, and displaying the sample input quantity to a user.

First claim

What is claimed is:

1. A method for quantifying a sample mutant nucleic acid, the method comprising:
determining a first reference threshold cycle for a first predetermined input quantity for a reference nucleic acid;
determining a first target threshold cycle for a first predetermined input quantity for a mutant nucleic acid;
determining a second reference threshold cycle for a second predetermined input quantity for the reference nucleic acid;
determining a second target threshold cycle for a second predetermined input quantity for the mutant nucleic acid;
calculating an inherent delta Ct between the threshold cycles of the first and second reference threshold cycles and the first and second target threshold cycles;
receiving a sample threshold cycle;
determining a mutant percentage of the sample based on the first and second reference threshold cycles and the first and second mutant threshold cycles, the inherent delta Ct and the sample threshold cycle using the equation:
Mutant percentage=0.5 ((Ct mu −Ct rf )−ΔCt inherent ) *100%;

and
displaying the mutant percentage to a user.

2. The method of claim 1 , wherein the first and second reference thresholds are determined by a gene reference assay.

3. The method of claim 1 , wherein the sample input quantity is calculated with the following equation:
sample input quantity %=2 −(ct mu −ct rf ) .

4. The method of claim 2 , wherein the sensitivities of the mutation detection assay and the gene reference assay are different.

5. The method of claim 2 , wherein the efficiencies of the mutation detection assay and the gene reference assay are different.

6. A system for quantifying a nucleic acid, the system comprising:
a threshold cycle determination processor comprising program code comprising instructions, which when executed cause the processor to:
determine a first reference threshold cycle for a first predetermined input quantity for a reference nucleic acid,
determine a first target threshold cycle for a first predetermined input quantity for a mutant nucleic acid,
determine a second reference threshold cycle for a second predetermined input quantity for the reference nucleic acid, and
determine a second target threshold cycle for a second predetermined input quantity for the mutant nucleic acid;
a quantity determination processor comprising program code comprising instructions, which when executed cause the processor to:
calculate an inherent delta Ct between the threshold cycles of the first and second reference threshold cycles and the first and second target threshold cycles;
receive a sample threshold cycle,
determine a mutant percentage of the sample based on the first and second reference threshold cycles and the first and second mutant threshold cycles, the inherent delta Ct and the sample threshold cycle using the equation:
Mutant percentage=0.5 ((Ct mu −Ct rf )−ΔCt inherent ) *100%;

and
a display screen configured to display the mutant percentage to a user.

7. The system of claim 6 , wherein the first and second reference thresholds are determined by a gene reference assay.

8. The system of claim 6 , wherein the sample input quantity is calculated with the following equation:
sample input quantity %=2 −(ct mu −ct rf ) .

9. The system of claim 7 , wherein the sensitivities of the mutation detection assay and the gene reference assay are different.

10. The system of claim 7 , wherein the efficiencies of the mutation detection assay and the gene reference assay are different.

Assignees

• Life Technologies Corp

Inventors

• Wu Yalei
• Keys David

Classifications

• C12Q1/6851 Primary

  Quantitative amplification · CPC title

• C12Q2537/16

  Assays for determining copy number or wherein the copy number is of special importance · CPC title

• C12Q2545/114

  involving a quantitation step · CPC title

• G16B45/00

  ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title