Method for the qualitative evaluation of real-time PCR data

Patent metadata

Field	Value
Publication number	US-12525322-B2
Application number	US-202017757464-A
Country	US
Kind code	B2
Filing date	Dec 18, 2020
Priority date	Dec 20, 2019
Publication date	Jan 13, 2026
Grant date	Jan 13, 2026

Abstract

A method is used for the qualitative evaluation of real-time PCR data, where a time/PCR amplification plot of an associated sample is classified as a negative plot or as a positive plot. The method involves providing a real-time PCR amplification plot to be classified, plotting at least 20 successive amplitude values of corresponding successive PCR cycle indices of the sample. Next, a quality metric is determined, on the basis of the at least one amplitude value. A first criterion is determined by a comparison of the quality metric with a first standard value. A sequence of values is then determined, which indicates a gradient of the PCR amplification plot to be classified, and a second criterion is determined as to whether the sequence of values exceeds a second standard value. Finally, the real-time PCR amplification plot is classified as a positive plot if all the criteria given above are satisfied.

First claim

The invention claimed is:

1 . A computer-implemented method for improving real-time PCR by the qualitative evaluation of real-time PCR data (DT), wherein a real-time PCR amplification curve (K 1 ) of an associated sample (P) is classified as a negative curve or as a positive curve, the method comprising:
providing real-time PCR reagents including an associated sample (P) including DNA having a target sequence, primers, DNA polymerase, free nucleotides and DNA-intercalating fluorescent pigments or sequence-specific fluorescence-labeled DNA probes; conducting real-time PCR cycles each so as to copy said target sequence by denaturing, primer hybridization and elongation in the associated sample (P); carrying out successive said PCR cycles of the associated sample (P); optically measuring fluorescence intensity at the end of each of said PCR cycles; providing the real-time PCR amplification curve (K 1 ) of the associated sample (P), wherein the real-time PCR amplification curve (K 1 ) has at least 20 chronologically successive amplitude values of corresponding chronologically successive PCR cycle indices of the associated sample (P), determining a quality metric (RG), which indicates a similarity of the real-time PCR amplification curve (K 1 ) to a straight line (GER), as a quality metric of a linear regression with respect to the real-time PCR amplification curve (K 1 ), wherein the determining of the quality metric (RG), is on a basis of the at least 20 chronologically successive amplitude values of corresponding chronologically successive PCR cycle indices, determining a first criterion by checking whether the quality metric (RG) of the linear regression falls below a first predefined value (VW 1 ), determining a value sequence (MK 1 ′), which indicates a slope of the real-time PCR amplification curve (K 1 ), and determining a second criterion as to whether the value sequence (MK 1 ′) exceeds a second predefined value (VW 2 ), and classifying the real-time PCR amplification curve (K 1 ) of the associated sample (P) as a positive curve in the case that the first criterion and the second criterion are met or as a negative curve if not met, wherein when the real-time PCR amplification curve (K 1 ) is classified as a positive curve this is indicative of a presence of the DNA having the target sequence in the real-time PCR and that the real-time PCR reagents functioned properly and were correctly measured, and wherein when the real-time PCR amplification curve (K 1 ) is classified as a negative curve this is indicative of no presence of the DNA having the target sequence in the real-time PCR, or that the real-time PCR reagents did not function properly or were not correctly measured.

2 . The computer-implemented method as claimed in claim 1 , further comprising:
determining a third criterion as to whether the value sequence (MK 1 ′), which indicates a slope of the real-time PCR amplification curve, exceeds a third predefined value (VW 3 ), and classifying the real-time PCR amplification curve (K 1 ) of the associated sample (P) as a positive curve in the case that the third criterion is met.

3 . The computer-implemented method as claimed in claim 1 , further comprising:
providing a real-time PCR amplification curve (P 1 ) of a positive control sample (PK) and a real-time PCR amplification curve (N 1 ) of a negative control sample (NK), determining a first secondary criterion as to whether a last fluorescence value of the real-time PCR amplification curve (P 1 ) of the positive control sample (PK) has exceeded a minimum value (MIW), determining a second secondary criterion as to whether a last fluorescence value of the real-time PCR amplification curve (N 1 ) of the negative control sample (NK) has not exceeded a maximum value (MAW), classifying the real-time PCR amplification curve (K 1 ) of the associated sample (P) as not valid if at least one of the first secondary criterion and the second secondary criterion is not met.

4 . The computer-implemented method as claimed in claim 1 ,
wherein the quality metric (RG) is determined as a quality metric of a linear regression with respect to the real-time PCR amplification curve (K 1 ), and wherein the first criterion is determined by checking whether the quality metric (RG) of the linear regression falls below the first predefined value (VW 1 ).

5 . The computer-implemented method as claimed in claim 1 , further comprising:
determining a last fluorescence value (LFL) of the real-time PCR amplification curve (K 1 ), and classifying the real-time PCR amplification curve (K 1 ) of the associated sample (P) as a positive curve as a function of the last fluorescence value (LFL) of the real-time PCR amplification curve (K 1 ).

6 . The computer-implemented method as claimed in claim 1 , comprising:
providing a device (V) for qualitative evaluation of real-time PCR data, including: a memory for providing the real-time PCR amplification curve (K 1 ) of an associated sample (P) having the at least 20 chronologically successive amplitude values of corresponding chronologically successive PCR cycle indices of an associated sample (P), and a processor for determining the quality metric (RG), which indicates a similarity of the real-time PCR amplification curve (K 1 ) to a straight line (GER), as a quality metric of a linear regression with respect to the real-time PCR amplification curve (K 1 ), on a basis of the at least 20 chronologically successive amplitude values of corresponding chronologically successive PCR cycle indices, for determining the first criterion by checking whether the quality metric (RG) of the linear regression falls below the first predefined value (VW 1 ), and for determining the value sequence (MK 1 ′), which indicates a slope of the real-time PCR amplification curve (K 1 ), and for determining the second criterion as to whether the value sequence (MK 1 ′) exceeds a second predefined value (VW 2 ), wherein the processor classifies the real-time PCR amplification curve (K 1 ) of the associated sample (P) as a negative curve or as a positive curve, and wherein the processor classifies the real-time PCR amplification curve (K 1 ) of the associated sample (P) as a positive curve in the case that the first criterion and the second criterion are met.

7 . The computer-implemented method (V) according to claim 6 , wherein the quality metric (RG) is a single quality metric.

8 . The computer-implemented method according to claim 7 , wherein the quality metric (RG) is a single quality metric.

9 . The computer-implemented method as claimed in claim 6 ,
wherein the memory is a digital storage medium; and wherein the processor is a computer processor, a graphics processor, a computer, a computer system, an application-specific integrated circuit, an integrated circuit, a one-chip system, a programmable logic element, or a field-programmable gate array having a microprocessor.

10 . The computer-implemented method as claimed in claim 9 , wherein the digital storage medium is a floppy disk, a DVD, a Blu-ray disk, a CD, a ROM, a PROM, an EPROM, an EEPROM, a FLASH memory, a hard drive, or another magnetic or optical memory.

11 . The computer-implemented method as claimed in claim 1 ,
wherein the first criterion is met if the quality metric (RG) falls below the first predefined value (VW 1 ), and wherein the second criterion is met if at least one value of the value sequence (MK 1 ′) exceeds the second predefined value (VW 2 ).

12 . The computer-implemented method according to claim 1 , wherein the quality metric (RG) is a single quality metric.

13 . The co

Assignees

• Euroimmun Medizinische Labordiagnostika Ag

Inventors

• Filippis Christodoulos
• Steller Ulf
• Schilling Fiona

Classifications

• C12Q1/6851

  Quantitative amplification · CPC title

• G16B25/20

  Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• G16H50/00

  ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics · CPC title

• G16B40/10 Primary

  Signal processing, e.g. from mass spectrometry [MS] or from PCR · CPC title