Primepcr ddpcr mutation detection assay kit

The ddPCR assays were performed with the PrimePCR™ ddPCR™ Mutation Detection Assay kit and PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories) (Additional file 1: Table S1). We used cfDNA from Multiplex I cfDNA Reference Standards (Horizon Discovery) that included wild-type cfDNA with mutant allele frequencies of 5%, 1%, and 0.1%. cfDNA Reference Standards (Horizon Discovery) with 0.1% mutant allele was serially diluted to wild-type cfDNA for analytical sensitivity of the ddPCR assay (Additional file 1: Table S3). Healthy control samples and DNA-free samples were also analyzed (Additional file 1: Table S2) [25 (link), 26 (link)]. Amplifications were carried out in a reaction volume of 20 μL on a QX100 Droplet Digital PCR System (Bio-Rad). The 20 μL PCR mix was composed of 10 μL Bio-Rad Super mix TaqMan, 1–2 μL of each amplification primer/probe mix, and 8–9 μL NAs. Thermal cycling comprised an initial denaturing and polymerase hot-start activating step of 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s and 55 °C for 60 s. Results were analyzed with QuantaSoft v.1.7.2 software (Bio-Rad) and reported as copies per milliliter of plasma.

ddPCR assays were performed with the Prime-PCR™ ddPCR™ Mutation Detection Assay Kit (Bio-Rad, Hercules, CA, USA) using an amplicon of 57 nt. DNA from the SW480 cell line was used as a positive control and DNA from leukocytes of a healthy donor served as a negative control. Background from water added to the reaction mixture instead of DNA was analyzed. This study was performed on a QX200 Droplet Digital PCR System (Bio-Rad), consisting of a C1000 Touch Thermalcycler, a QX200 Droplet Generator, and a QX200 Droplet Reader. The PCR reaction mixture (20 µL) contained 10 µL of ddPCR Supermix (no dUTP) for probes, 1 µL of each primer/probe mix (target and reference, labeled with HEX and FAM fluorophores, respectively), and 8 µL of plasma-extracted DNA. A total amount of 130 ng of DNA was added per well in case of positive and negative controls and in DNA extracted from tumors. The thermal cycling started with 10 min at 95 °C, followed by 40 cycles of 94 °C for 30 s and 55 °C for 60 s. Results were analyzed using Quantasoft v.1.7 software (Bio-Rad) and reported as copies per mL of plasma or % of mutant DNA in the tumor. Four replicates of each sample were analyzed.

ddPCR assays were performed with the PrimePCR™ ddPCR™ Mutation Detection Assay kit and PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories, Hercules, CA, USA) (Additional file 1: Table S1). We selected three hotspot mutations of Ex19del, L858R, and T790M. L858R and Ex19del are the most common forms of EGFR sensitizing mutations (85%) that are responsive for EGFR TKI treatment [23 (link)]. In case of progression on 1st generation TKI treatment, T790M mutation testing is recommended as acquired T790M mutation is the most common resistance mechanism (> 50%) that is responsive for 3rd generation TKI treatment [23 (link), 24 (link)]. The limitation of detection (LOD) was determined as the lowest mutant concentration above the 95% confidence interval (CI) of the wild-type (WT) control, which was determined using a Poisson model (Additional file 1: Table S2). Validation ddPCR was performed using Multiplex I cfDNA Reference Standard (Horizon Discovery, Cambridge, UK) (Additional file 1: Table S3). The ability to detect EGFR mutation based on type of input nucleic acid (short-length exoTNA and a size range of exoDNA) was evaluated (Additional file 1: Figures S1 and S2). Analytic performance of isolated cfDNA and short-length exoTNA was evaluated using ddPCR. We assessed the influence on cfDNA levels and short-length exoTNA according to storage period.