Detection of MegaTAL-induced HIV pol Mutation By Droplet Digital PCR

Thesis (Master's)--University of Washington, 2015

Reliable detection of low-level mutations in HIV-1 provirus is still a challenge. Current methods regularly used in laboratories, Surveyor assay and bulk sequencing, only have a limit of detection about 10 ~ 20%. In the Jerome lab, a potential cure for HIV, megaTALs, is under development and a method to evaluate its outcome is in need. In this thesis, a droplet digital PCR assay that can detect low-level target mutations in HIV provirus induced by megaTALs was developed. This assay was validated with both plasmid and cell line samples. This assay had demonstrated high analytical linearity (r2 > 0.99) and repeatability (CVs < 7%). The Limit of Blank of this assay is 0.56%. The Limit of Detection of this assay is 1.06%. The Limit of Quantitation of this assay is 2.19%. This assay showed higher accuracy than Surveyor assay. The results of the same sample generated by this assay were comparable to those of clonal sequencing and Illumina sequencing. This assay has approved that ddPCR can be use as a reliable mutation detection method in HIV with high precision and accuracy.