Methods and systems for validation of a nucleic acid amplification assay
Inventors
Phillips, Cynthia L. • Bramwell, Kenneth K. C. • Ririe, Kirk M. • Poritz, Mark Aaron
Assignees
Publication Number
US-12291745-B2
Publication Date
2025-05-06
Expiration Date
2039-01-29
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Abstract
Systems, methods, and apparatus are provided for external control testing of an assay system.
Core Innovation
The invention provides systems, methods, and apparatus for external control testing of nucleic acid amplification assays, especially multiplexed PCR. It centers on the creation and use of external control material (ECM) comprising a set of positive control sequences that are engineered to minimize the likelihood of false positive results. Each positive control sequence is designed to be amplified by the same primer set as its corresponding test sequence, but the sequence includes an engineered region that is detectably distinguishable, such as by having a shifted melting temperature, allowing differentiation between true positives and control-derived positives.
The problem addressed is the risk of contamination and false positive results that occur when clinical laboratories and other users employ organism- or nucleic acid-based positive controls. Traditional approaches have required manipulation of live or inactivated pathogens, which can be hazardous, cause carryover contamination, and generate indistinguishable results from true positives. This is particularly problematic for rare or high-risk pathogens, where obtaining or managing control material poses significant safety and practicality issues.
To solve this, the invention describes positive control sequences engineered to be distinguishable from the test amplicons, often through melt temperature differences of at least 2–10°C. These controls are provided in formats such as dried samples in vials or swabs, stable at room temperature, and designed to be introduced into multiplex PCR assay systems without the presence of test organism or sequence. This allows robust, safe, contamination-minimized, and easy-to-use assay validation and quality control applicable to a wide variety of multiplexed nucleic acid amplification environments.
Claims Coverage
The patent contains two independent claims that focus on methods for quality control in multiplexed PCR systems using specifically engineered positive control materials.
Engineered positive control sequences distinguishable by melt temperature
The method involves providing a positive control material with a plurality of positive control sequences, each corresponding to a test sequence, where each positive control sequence comprises the same forward and reverse primer binding sites as its corresponding test sequence but includes an engineered sequence (between the primers) that is different from the native sequence. Each positive control amplicon has a melting temperature that is detectably different and distinct (about 2–10°C higher or lower) from its corresponding test amplicon, ensuring that the controls are distinguished via melting analysis.
Multiplex assay validation for diverse infectious disease targets using engineered controls
A method for quality control of a multiplexed PCR system covering a broad range of disease assays (such as Anthrax, Leptospirosis, Plague, Ebola, Malaria, etc.) by introducing a specifically engineered positive control, where each control sequence is designed to be amplified by the same primers as the corresponding pathogen but contains an engineered segment between primer sites to differentiate via melting analysis. The method includes detecting and verifying positive control amplicons, with each such amplicon melting at a temperature distinct from native pathogen amplicons.
The inventive features protect the method of using engineered positive control sequences for multiplexed PCR quality control, specifically by differentiating positive controls from test sequences through melt temperature, and enabling safe and practical external assay validation across a range of infectious disease targets.
Stated Advantages
Minimizes risk of false positive results due to contamination from positive control materials by making control sequences distinguishable from test amplicons.
Does not require handling or propagation of live or inactivated pathogens, eliminating biological hazards and obviating the need for high biosafety containment.
Allows room temperature storage and stability of the external control material, simplifying shipping and logistical requirements.
Enables assay validation and quality control without the need for highly trained personnel, sample preparation, or aliquoting of standards.
Detection of positive control material and pathogens is mutually exclusive, so positive control contamination does not cause false positives in clinical samples.
Facilitates rapid, simple, and contamination-minimized assay validation in clinical and non-clinical laboratories, including resource-limited and point-of-care settings.
Documented Applications
Laboratory quality control and validation of multiplexed PCR assays detecting various pathogens, including for clinical diagnostic laboratories.
External validation of in vitro diagnostic (IVD) systems, including verification of assay and instrument performance for new or repaired instruments and new users.
Verification of multiplex panel assays for a broad range of infectious diseases, such as anthrax, leptospirosis, plague, tularemia, typhoid fever, malaria, leishmaniasis, chikungunya fever, Crimean-Congo hemorrhagic fever, dengue fever, Ebola virus, Marburg virus, Lassa fever, West Nile fever, yellow fever, and Zika virus.
Use in non-clinical laboratory industries that routinely run control materials for nucleic acid-based testing.
Initial laboratory verification, assay validation, operator training, new shipment/lot verification, and after instrument maintenance of multiplexed PCR platforms such as the FilmArray Global Fever Panel.
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