Browsing by Subject "Droplet Digital PCR"
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Item The Effects of Known PCR Inhibitors on Droplet Digital PCR Performance(2015-05-01) Kolar, William Patrick; Joseph E. Warren; Michael Allen; Harlan P. JonesThis study is an analysis of how forensic samples may be affected by compounds that can inhibit the polymerase chain reaction (PCR). Inhibitors associated with forensic samples include substances that are endogenous with the sample (calcium, melanin), absorbed from the environment (humic acid, indigo dye)1, or introduced during laboratory processing (phenol1 , glove powder2). Human DNA quantification kits using quantitative real-time PCR (qPCR) instrumentation, the current standard for the quantification of DNA, are used to detect inhibitors in samples. Droplet digital PCR (ddPCR) relies on absolute quantification of positive and negative droplets and therefore may be less susceptible to inhibition than traditional qPCR techniques. Samples were prepared with a range of known inhibitor concentrations including aluminum (Al), copper (Cu), iron (Fe), and humic acid (HA). Metal ion concentrations ranged from 7 mM to 43 mM and Humic acid concentrations ranged from 166.67 ng/μL to 1000 ng/μL. Duplicate samples with 5 ng/μL of control DNA were used to determine the robustness of ddPCR in the presence of with inhibitors using the QX100 ddPCR system (BIO-RAD, Hercules, CA). Results indicate that iron and humic acid both show little effect on ddPCR: iron shows inhibition only with extreme inhibitor concentration. By contrast, copper slowly drops the amplitude of the positive droplets to the baseline with increasing inhibition. Aluminum drastically impacts ddPCR performance, resulting in positive droplets up to six times the amplitude of normal droplets in a scattered pattern, making the results unusable. These findings suggest that ddPCR may prove to be a more robust technique for inhibited forensic samples in some but not all settings.Item Validation of Droplet Digital PCR (ddPCR) for the Detection and Absolute Quantification of Borrelia DNA in Ixodes Ticks(2015-05-01) King, Jenny L.; Michael Allen; Joseph E. Warren; Raghu R. KrishnamoorthyIn this research, QX200 Droplet Digital PCR (ddPCRTM) system protocols for the detection of bacterial (Borrelia burgdorferi and Borrelia miyamotoi) DNA were developed and tested. Existing Ixodes scapularis samples collected from Cape Cod, Massachusetts and previously determined to be 60% positive for B. burgdorferi were utilized to investigate absolute bacterial genome carriage per tick using the ddPCR assays optimized here. The ddPCR technology proved to be a reliable means for detection and absolute quantification of control bacterial DNA with sensitivity as low as 10 spirochetes per μl input DNA. Application of ddPCR revealed an average B. burgdorferi carriage level of 27,239 copies in infected ticks (range: 231- 118,407 copies), 2,197 copies in infected nymphs (range: 231- 4,983 copies), and 45,620 copies in infected adults (range: 5,647- 118,407 copies). This is the first known and validated application of ddPCR for the detection of Borrelia DNA in Ixodes ticks.