	US 12,291,745 B2
	Methods and systems for validation of a nucleic acid amplification assay
Cynthia L. Phillips, Salt Lake City, UT (US); Kenneth K. C. Bramwell, Salt Lake City, UT (US); Kirk M. Ririe, Salt Lake City, UT (US); and Mark Aaron Poritz, Salt Lake City, UT (US)
Assigned to Biofire Defense, LLC, Salt Lake City, UT (US)
Appl. No. 16/965,853
Filed by BIOFIRE DEFENSE, LLC, Salt Lake City, UT (US)
PCT Filed Jan. 29, 2019, PCT No. PCT/US2019/015510 § 371(c)(1), (2) Date Jul. 29, 2020, PCT Pub. No. WO2019/152336, PCT Pub. Date Aug. 8, 2019.
Claims priority of provisional application 62/623,802, filed on Jan. 30, 2018.
Prior Publication US 2023/0159998 A1, May 25, 2023
Int. Cl. C12Q 1/68 (2018.01); B01L 3/00 (2006.01); B01L 7/00 (2006.01); C12Q 1/686 (2018.01)

CPC C12Q 1/686 (2013.01) [B01L 3/502715 (2013.01); B01L 7/52 (2013.01); B01L 2200/16 (2013.01); B01L 2300/0654 (2013.01); B01L 2300/0861 (2013.01); B01L 2300/18 (2013.01); B01L 2400/0481 (2013.01); C12Q 2600/16 (2013.01); C12Q 2600/166 (2013.01)]

12 Claims

1. 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.