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US 12,291,745 B2Granted
Methods and systems for validation of a nucleic acid amplification assay
AssigneeBioFire Defense, LLC
InventorsCynthia L. Phillips, Kenneth K. C. Bramwell, Kirk M. Ririe, Mark Aaron Poritz
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Abstract
Systems, methods, and apparatus are provided for external control testing of an assay system.
Claims12
A method of quality control of a multiplexed PCR system using a positive control material comprising the steps of:
- providing the positive control material comprising a plurality of positive control sequences corresponding to a plurality of test sequences,
- providing an assay device comprising a first reaction chamber provided with a plurality of primers for multiplexed nucleic acid amplification of the plurality of positive control sequences,
- wherein each positive control sequence comprises the same forward and reverse primer binding sites as its corresponding test sequence and an engineered sequence between the forward and reverse primer binding sites that is different than its corresponding test sequence,
- introducing the positive control material into the assay device, wherein the introducing does not include introducing a test sequence with the positive control material into the assay device,
- amplifying the plurality of positive control sequences in the first reaction chamber with the plurality of primers to yield a plurality of positive control amplicons,
- detecting the positive control amplicons,
- melting each positive control amplicon, and
- verifying by melting that each amplified positive control amplicon represents a positive control sequence,
- wherein detecting each of the positive control amplicons indicates that the multiplexed PCR system is operating correctly, and
- wherein each amplified positive control sequence has a melting temperature that is detectably different and distinct from a melting temperature of its corresponding test sequence as a result of the engineered sequence in each positive control sequence between the forward and reverse primer binding sites, and
- wherein each positive control sequence melts in a melt window at a temperature in a range of about 2-10° C. higher or lower than a melt window of its corresponding test sequence.
8 more elements
A method of quality control of a multiplexed PCR system using a positive control material comprising the steps of:
- providing an assay device comprising a first reaction chamber provided with a plurality of primers for multiplexed nucleic acid amplification,
- providing a positive control material comprising a plurality of control sequences configured to be amplified by the plurality of primers, wherein each one of the plurality of control sequences comprises a same forward and a same reverse primer binding site as a corresponding test nucleic acid sequence and an engineered sequence between the forward and reverse primer binding sites that is different than the corresponding test nucleic acid sequence, wherein the plurality of control sequences control for amplification of one of more of Anthrax, Leptospirosis, Plague (Yersinia pestis), Tularemia, Typhoid fever, Paratyphoid fever, Malaria, Leishmaniasis, Chikungunya fever, Crimean-Congo Hemorrhagic Fever, Dengue fever, Ebola virus, Marburg virus, Lassa fever, West Nile fever, Yellow fever, or Zika fever,
- introducing the positive control material into the assay device,
- amplifying the plurality of positive control sequences in the first reaction chamber with the plurality of primers to yield a plurality of positive control amplicons, and
- detecting the positive control amplicons,
- melting each positive control amplicon, and
- verifying by melting that each amplified positive control amplicon represents a positive control sequence,
- wherein detecting each of the positive control amplicons indicates that the multiplexed PCR system is operating correctly, and
- wherein each amplified positive control sequence has a melting temperature that is detectably different and distinct from a melting temperature of each corresponding test sequence as a result of the engineered sequence in each positive control sequence between the forward and reverse primer binding sites.
6 more elements
Dependencies are inferred from the claim text.