Absolute Quantification with the LightCycler® Carousel-Based System
The Absolute Quantification module uses external standards to calculate an absolute concentration value for an unknown sample. LightCycler® Software 4.1, 4.05 and 3.5.3 allow you to run a standard curve within your experiment or to import a previously generated standard curve.
LightCycler® Software Versions 4.1, 4.05 and 3.5.3 offer two absolute quantification methods:
1. Automated Method / Second Derivative Maximum Method
The automated method identifies the crossing point of a sample, i.e. where the fluorescence curve of the sample turns sharply upward. This turning point will be a maximum on the second derivative plot of the reaction.
This method allows you to use internal controls.
2. Fit Points Method
The Fit Points method converts the exponential fluorescence curve of a sample into a straight line (a linear curve). The beginning of this line is extrapolated until it intersects a horizontal line called the crossing line. The intersection of these two lines is defined as the crossing point for the sample. The software uses the calculated crossing points of the standards to generate a standard curve (crossing point versus sample concentration).
The Fit Points method requires you to perform most of the analysis steps manually; it does not allow you to use internal controls.
For more information, see Related Documents.
Absolute Quantification with External Standards
LightCycler® - FastStart DNA Master HybProbe has been used to amplify a segment within the repetitive insertion sequence (IS481) of Bordetella pertussis. Incorporation of a species-specific region in the design of the primers and hybridization probes guaranteed strain-specific detection. In addition IS481 is a very sensitive target, due to its high copy number per cell.
- Standards: Bordetella pertussis DNA was isolated from cultured strains, using the High Pure PCR Template Preparation Kit. Concentrations ranging from a 1:10 dilution (0.1 µg/sample = approx. 5 x 107 organisms) down to a 1:1,000,000 dilution (1 pg/sample = approx. 500 organisms) were used to generate a standard curve (sample number 1 – 6).
- Sample 7 is the no template control (NTC)
- Unknowns: DNA prepared from nasopharyngeal research specimens obtained from B. pertussis-positive subjects.
Result: For the three samples the amount of B. pertussis were determined to be 144,200 pg/sample (= 7 x 106 organisms); 11,520 pg/sample (= 6 x 105 organisms); and 4,918 pg/sample (= 2 x 105 organisms).
Note: The bacterium Bordetella pertussis is the causative agent of whooping cough, which is an infectious disease occurring worldwide with a high incidence among young, unvaccinated infants