Increasing Multiplex Level by Externalization of Passive Reference in PCR Reactions

• US 20130040843A1
• Filed: 02/04/2011
• Published: 02/14/2013
• Est. Priority Date: 02/05/2010
• Status: Active Grant

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Inventors: Stropp, Udo, Von Terne, Christian
• Granted Patent: US 9,506,112 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Methods for increasing multiplex level by externalization of a passive reference in polymerase chain reactions (PCR) are provided. An exemplary method comprises providing a first mastermix including a passive fluorescence dye in at least a first well of a plate; providing a second mastermix including an active fluorescence dye in at least a second well of the plate; wherein the passive fluorescence dye and the active fluorescence dye emit a same spectrum and an intensity of the spectrum is adapted to be measured; and wherein the first mastermix is devoid of an active fluorescence dye emitting the same spectrum and the second mastermix is devoid of the passive fluorescence dye emitting the same spectrum. Numerous other aspects are provided.

Claims

1. A method comprising:

providing a plate including a plurality of wells;

providing a first mastermix including a passive fluorescence dye in at least a first well of the plate;

providing a second mastermix including an active fluorescence dye in at least a second well of the plate;

wherein the passive fluorescence dye and the active fluorescence dye emit a same spectrum and an intensity of the spectrum is adapted to be measured; and

wherein the first mastermix is devoid of an active fluorescence dye emitting the same spectrum and the second mastermix is devoid of the passive fluorescence dye emitting the same spectrum.

2. The method of claim 1 further comprising:

providing the mastermix including the active fluorescence dye in two or more of the plurality of wells.

3. The method of claim 1 further comprising:

performing a polymerase chain reaction in each of the first and second wells.

4. The method of claim 3 further comprising:

measuring an intensity of the passive and active fluorescence dyes for each cycle of the polymerase chain reaction.

5. The method of claim 4 further comprising:

providing data of the measured intensity of the passive and active fluorescence dyes for each cycle of the polymerase chain reaction.

6. The method of claim 5 further comprising:

extracting well-wise information from the data for the second well containing the active fluorescence dye.

7. The method of claim 6 wherein extracting well-wise information further comprises:

detecting a background region of the data for the second well.

8. The method of claim 7 further comprising:

determining a slope, level and magnitude of noise for the background region data for the second well.

9. The method of claim 8 further comprising:

extracting plate-wise information from the data for each well containing the passive fluorescence dye.

10. The method of claim 9 further comprising:

combining the well-wise information and plate-wise information to form a virtual reference.

11. The method of claim 10 further comprising:

normalizing the data from the wells including the mastermix with the active fluorescence dye with the virtual reference.

12. A method comprising:

providing a first mastermix including a passive fluorescence dye in at least a first well of a plate;

providing a second mastermix including two or more active fluorescence dyes in at least a second well of the plate;

wherein the passive fluorescence dye emits a same spectrum as one of the active fluorescence dyes and an intensity of the dye is adapted to be measured; and

wherein the first mastermix is devoid of an active fluorescence dye emitting the same spectrum and the second mastermix is devoid of the passive fluorescence dye emitting the same spectrum.

13. The method of claim 12 further comprising:

providing the mastermix including the two or more active fluorescence dyes in two or more of the plurality of wells.

14. The method of claim 12 further comprising:

performing a polymerase chain reaction in each of the wells.

15. The method of claim 14 further comprising:

measuring an intensity of the passive and active fluorescence dyes for each cycle of the reaction.

16. The method of claim 15 further comprising:

providing data of the measured intensity of the passive and active fluorescence dyes for each cycle of the polymerase chain reaction.

17. The method of claim 16 further comprising:

extracting well-wise information from the data for each well containing the active fluorescence dye.

18. The method of claim 17 wherein extracting well-wise information further comprises:

detecting a background region in the data from the second well; and

determining a slope, level and magnitude of noise in the background region.

19. The method of claim 18 further comprising:

extracting plate-wise information from the data for each well containing the passive fluorescence dye.

20. The method of claim 19 further comprising:

combining the well-wise information and plate-wise information to form a virtual reference.

21. The method of claim 20 further comprising:

normalizing the data of the same-dye in the wells including the mastermix with the active fluorescence dye with the virtual reference.

22. The method of claim 20 further comprising:

normalizing the data of the two or more active dyes of the wells including the mastermix with the two or more active fluorescence dyes with the virtual reference.