Although nucleic acid tests (NATs) for human immunodeficiency virus (HIV) exhibit many advantages, such as early detection and viral load quantification, over immunological assays, their widespread use is limited by their demand for high-level infrastructure, sophisticated equipment, and advanced staff competence. Furthermore, when quantifying viral loads of patients, it has been reported that these assays can underestimate viral quantities by 22- to 100-fold due to primer-template mismatches in more divergent HIV subtypes. Therefore, we have developed a cost-effective and sequence-independent assay for the detection and quantification of HIV utilizing a modified nucleic acid sequence-based amplification (NASBA) protocol coupled to an electrochemical DNA biosensor. The modified NASBA reaction involves the addition of a 22-nucleotide tag between the T7 RNA Polymerase Promoter and the hybridizing region of the reverse primer. As a result, this tag will be placed at the 5' end of each amplicon regardless of the target sequence. We then designed the DNA sensor to hybridize to this segment of the amplicon specifically. Therefore, hybridization, and subsequently, detection, is dependent only on the presence of this tag and not the viral RNA sequence itself. As a result, the issue of underestimating viral loads is eliminated as multiple primers can be used in one reaction without having to use multiple sensors. The use of an isothermal NASBA technique and a reusable gold-disc electrode for the sensor helps drive down the cost of the assay by eliminating the need for thermocyclers and fluorometers used by conventional NATs.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses-1930 |
| Date | 01 January 2021 |
| Creators | El Merhebi, Omar |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Honors Undergraduate Theses |
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