Liquid biopsy is emerging as a powerful, cost effective tool for genotyping cancer cells, individualizing therapy to match a growing arsenal of drugs with cancer genetics, and monitoring genetic shifts in cancer cells in nearly real time. Liquid biopsy has the potential of significantly improving treatment outcomes and reducing costs by enabling selection of effective drugs and detection of the presence and evolution of drug-resistance. The detection of mutant alleles in bio samples, such as blood and urine often requires a ?needle in a haystack? approach since excess wild-type (WT) DNA exhausts essential reagents during polymerase amplification and masks mutation alleles' signals. In developed countries, cancer genotyping is carried out with sophisticated, expensive equipment such as digital PCR (ddPCR) and next generation sequencers, is laborious, and requires well-trained technicians. In low and middle income countries, such capabilities are either non-existent or limited to a few central laboratories that lack the capacity to meet the needs of a large and growing population of cancer patients, and are often located great distance from the patient. To address this critical need, an interdisciplinary team of US and Chinese investigators proposes an inexpensive, point-of-care smartphone- based system for detecting rare mutant alleles in body fluids. The proposed system builds on our prior work with minimally instrumented and un-instrumented molecular diagnostics. Our system accepts a raw sample, such as whole blood, processes the sample, and provides test results. Our system will include a sample enrichment step that utilizes DNA-guided cleaving enzymes of the Argonaute family to digest wild type (WT) alleles while sparing the mutant alleles of interest. This will be followed with a loop mediated isothermal amplification (LAMP) that utilizes peptide nucleic acid (PNA) clamp to selectively amplify mutant alleles, but not WT-alleles. Amplicons will be detected with bioluminescent reporters and a smartphone camera. A custom smartphone application will analyze the recorded signal; report test results; and, in the future, transmit these results to the patient's doctor and records and, in de-identified form, to the cloud for spatiotemporal surveillance, allowing public health officials identify hotspots. Our preliminary data indicates that our approach has high likelihood of success. Our program will also build capacity in China. We will initiate a new program on mobile diagnostics at the Beijing University of Chemical Technology (BUCT), catalyze collaborations between Chinese cancer researchers and engineers, train Chinese students and researchers in novel diagnostic assays and point of care devices, and establish the foundation to enable sustainability of this research program in China after the conclusion of this R21 research.
This project will develop an inexpensive, smartphone-based point-of-care system to detect mutant alleles in blood samples of cancer patients, enabling personalized therapy and monitoring of evolution of drug resistance.