STAR Technology
STAR (Scalable Target Amplification Routine) technology is a method for simultaneous quantitative measurement of multiple target nucleic acids. STAR technology is based on novel functional integration of polymerase chain reaction (PCR) and capillary electrophoresis (CE). In a typical STAR assay, amplification of multiple nucleic acid targets is monitored by sampling PCR reaction during sequential PCR cycles and separating, and quantifying, the PCR products by CE. From the data, amplification curves are constructed that are used for simultaneous quantification of multiple nucleic acid targets.
Similar to real-time PCR amplification, a threshold cycle Ct (cycle at which measured amount of PCR product crosses a pre-selected value) is determined from amplification curve for each of the targets in the exponential phase of PCR amplification. In contrast to real-time PCR, where standards in a separate reaction are used to establish a calibration curve to measure the Ct value for the assay targets, STAR uses multiple internal standards in each reaction to generate a precise, and reproducible, calibration curve for each individual assay.
Inhibition free multiplex amplification
Multiplex PCR can be adversely affected by interactions between multiple primers and amplicons present in PCR reaction. Specifically, the amplification of rare targets may be inhibited by the amplification of abundant targets in the same reaction. Quantitative multiplexed PCR can be particularly sensitive to this problem.
PrimeraDx’s proprietary solution to this problem is two-fold: First, we have developed software specifically for multiplex primer design. This software selects and compares the primers and amplicons to avoid negative interaction between any components of the reaction. Second, a proprietary reagent formulation, developed for use in highly multiplex PCR, mitigates primer competition problems. By combining these two novel approaches, PrimeraDx has enabled highly multiplexed nucleic acid target amplification.
Multiplexed assays, developed at PrimeraDx, undergo careful validation and verification to ensure that performance is comparable to single target real-time PCR, in terms of sensitivity, specificity, reproducibility, and if relevant, linearity and quantification accuracy.
Standardization
Quantitative multiplexed target amplification using STAR technology addresses several long standing issues in molecular diagnostics:
- STAR can ensure the quality of nucleic acid sample preparation by including external and internal controls that assess the recovery of target nucleic acids from the clinical specimen.
- STAR allows for verification of the measurement of each individual target by including target-specific positive controls, thus eliminating of the possibility of inference by other targets in the assay.
- STAR enables standardization of nucleic acid measurements through the incorporation of universal, chemically-defined DNA standards. These standards act as a common “measuring stick” for quantifying individual nucleic analytes. Standardization of nucleic acid detection and quantification is a key component in the effort to consolidate data from different investigators, laboratories and manufacturers and to create reproducible and comparable clinical information.
Platform
STAR assays are performed as an integrated process, in which PCR amplification takes place concurrently with CE analysis. This integrated approach has been embodied in the ICEPlex instrument system.