US 12,467,102 B2
Massively parallel COVID-19 diagnostic assay for simultaneous testing of 19200 patient samples
Howard Salis, University Park, PA (US); Alexander Reis, University Park, PA (US); and Ayaan Hossain, University Park, PA (US)
Assigned to The Penn State Research Foundation, University Park, PA (US)
Filed by THE PENN STATE RESEARCH FOUNDATION, University Park, PA (US)
Filed on Mar. 18, 2022, as Appl. No. 17/698,975.
Claims priority of provisional application 63/321,550, filed on Mar. 18, 2022.
Claims priority of provisional application 63/162,847, filed on Mar. 18, 2021.
Prior Publication US 2022/0307095 A1, Sep. 29, 2022
Int. Cl. C12Q 1/70 (2006.01); C12N 15/10 (2006.01)
CPC C12Q 1/701 (2013.01) [C12N 15/1065 (2013.01)] 17 Claims
 
1. A method for parallel detection, comprising:
obtaining a plurality of samples;
preparing a plurality of reverse transcription (RT) primers corresponding to the plurality of samples, wherein each RT primer of the plurality of RT primers comprises a sample-specific barcode comprising a sample-specific barcode sequence comprising random nucleotides, wherein no two RT primers have a common sample-specific barcode sequence and each sample-specific barcode sequence is distinguishable from the sample-specific barcodes of each other RT primer, based on a predetermined edit distance;
performing reverse transcription based on the plurality of samples and the plurality of RT primers;
generating a plurality of RT reaction products corresponding to the plurality of RT primers based on the performed reverse transcription, wherein each RT reaction product of the plurality of RT reaction products comprises a cDNA with a respective sample-specific barcode, the cDNA of each RT reaction product corresponding to a respective sample of the plurality of samples;
combining a portion of each RT reaction product of the plurality of RT reaction products in a single container to form a combined RT reaction product;
performing polymerase chain reaction (PCR) based on the combined RT reaction product;
generating a plurality sets of amplified cDNAs based on the performed PCR, the plurality sets corresponding to the plurality of RT reaction products, each amplified cDNA of the plurality sets of amplified cDNAs comprising the sample-specific barcode and a pool-specific barcode, the pool-specific barcode corresponding to the combined RT reaction product;
obtaining a plurality sets of sequencing reads based on the plurality sets of amplified cDNAs, the plurality sets of sequencing reads corresponding to the plurality sets of amplified CDNAs;
quantifying the plurality sets of sequencing reads; and
determine a diagnostic outcome of each sample of the plurality of samples based on the quantified plurality of sequencing reads,
wherein the plurality of samples comprises a sample number greater than 384 and less than or equal to 19,200.