<p>Loop-mediated Isothermal Amplification (LAMP) is a promising
technology to address diagnostic and surveillance testing during public health
crises, such as the current COVID-19 pandemic; however, primer design and assay
optimization remain a barrier to enabling rapid deployment of assays based on
LAMP. Herein, we introduce a design and screening process that allows for strategic
determination of optimally performing primer sets and standardized assay
conditions which enable execution of LAMP at point-of-care (POC) settings using
complex human samples such as saliva. A total of 20 primer sets targeting the
N, E, RdRP, and orf1ab genes of the SARS-CoV-2 were designed, screened, and selected
based on performance metrics such as reaction time, sensitivity, and
specificity. Of these 20 primer sets, only two primer sets (orf1ab.2 and orf1ab.4)
proved to be viable for use in the final assay. Colorimetric RT-LAMP of the
selected primer set, orf1ab.2 was shown to produce a distinct color change in
contrived samples containing heat-inactivated SARS-CoV-2 in 5% saliva. The
limit of detection of our assay using primer set orf1ab.2 was determined to be 1000
copies/µL of saliva
collected. Furthermore, methods are introduced which allow for the
high-throughput design of LAMP primers using standard software tools and the <i>in-silico</i>
performance of LAMP primer sets. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/14511612 |
| Date | 29 April 2021 |
| Creators | Josiah Levi Davidson (10723713) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY-ND 4.0 |
| Relation | https://figshare.com/articles/thesis/Design_and_Selection_of_RT-LAMP_Primer_Sets_Targeting_SARS-CoV-2_in_Complex_Human_Samples/14511612 |
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