Cervical cancer is a major problem in the developing world and low- resource settings where standard screening techniques are not accessible. Cervical cancer is one of the few cancers that can be successfully treated when detected early. Therefore, there exists a high clinical need to screen for cervical cancer early. The etiological agent of cervical cancer is the human papillomavirus (HPV), with 70% of cases related to HPV genotypes 16 and 18.
I sought to increase access to screening by developing a fully integrated and multiplexed molecular diagnostic assay to extract, amplify, detect, and distinguish HPV16 and HPV18 DNA on a low-cost paperfluidic platform for point- of-care (POC) applications. Isothermal (one temperature) loop-mediated amplification (LAMP) was used to amplify HPV DNA instead of the traditional polymerase chain reaction (PCR) that requires multiple temperatures. The amplified HPV16 and HPV18 DNA were differentially detected on a simple lateral flow strip – similar to that used in a pregnancy test – generating a visible
colorimetric readout for each specific genotype.
LAMP amplification is difficult to characterize and current methods were insufficient in providing specificity at the level needed for a multiplexed assay. Therefore, a novel characterization strategy was developed based on fluorescence to distinguish positive LAMP amplification products. This workflow used differential fluorescent tags to identify whether HPV16 DNA or HPV18 DNA was present and simplified complex nonspecific LAMP smears to a specific band pattern.
After singleplex HPV16 and HPV18 LAMP assays were optimized with the new workflow, the two singleplex assays were successfully combined into one multiplex reaction with 12 primers, a nontrivial feat. Each assay step – DNA extraction, amplification, and detection – was optimized and integrated into a single chip that can control the timing of each step. Several chip configurations were tested to determine the optimal chip form factor, and a small subset of clinical samples were tested to demonstrate feasibility in low-resource settings. With this diagnostic platform, asymptomatic patients positive for HPV16 DNA and HPV18 DNA can be screened more closely, allocating precious resources to those most at risk, a beneficial use in both low-resource settings and the USA.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/34408 |
| Date | 28 February 2019 |
| Creators | Wong, Winnie S. |
| Contributors | Klapperich, Catherine M. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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