Development of Rapid, Specific, and Field-Deployable Diagnostic Assays For Sheepox and Goatpox Viruses Using Crispr-Cas Technology
| dc.contributor.advisor | Dr. Olana Merera | |
| dc.contributor.advisor | Dr. Samson Leta | |
| dc.contributor.author | Birtukan Zenebe | |
| dc.date.accessioned | 2024-11-04T07:38:46Z | |
| dc.date.available | 2024-11-04T07:38:46Z | |
| dc.date.issued | 2024 | |
| dc.description | MSc Thesis | |
| dc.description.abstract | Ethiopia is home to a large population of sheep and goats, which are raised for their meat, hides, and skins. Even with this huge financial resource, animal diseases are still the main factor that limits their productivity. sheeppox and goatpox is one of the most prevalent diseases caused by a genus of Capripoxvirus that have a negative impact on sheep and goats throughout the world. One of the main obstacles to the quick diagnosis of the sheeppox and goatpox viruses is the lack of rapid, specific, and point-of-care diagnostic tests. Thus, the aim of this study is to develop a rapid, specific and field deployable diagnostic assay to detect sheeppox and goatpox viruses based on recombinase polymerase amplification (RPA) and trans-cleavage activity of clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) and to validate its diagnostic specificity. Thus, a lab-based experimental study was employed from November 2023 to April 2024 to develop this assay. RPA-CRISPR/Cas12a-lateral flow assay was developed to detect the entry fusion complex component gene of the sheeppox and goatpox viruses’ genomes. The assay was optimized to be completed within one hour, including nucleic acid extraction for 25 minutes, RPA reaction at 39°C for 15-20 minutes, CRISPR/Cas12a reaction at 37°C for 20 minutes, and visual detection through lateral flow (3 minutes). The primers for RPA, CRISPR RNA (crRNA), and ssDNA reporter for lateral flow detection (LFD) were synthesized and optimized. In this study, quick, specific, and versatile diagnostic method for goatpox and sheeppox virus was successfully developed. This innovative approach harnesses recombinase-polymerase amplification in conjunction with the CRISPR/Cas12a system, offering a cost-effective and efficient solution for identifying these viral infections. This diagnostic technique shows no cross-reactivity with Peste des petits ruminants (PPR). In conclusion, RPA-CRISPR/Cas12a is a potentially useful method for quickly and accurately diagnosing goatpox and sheeppox, especially in environments with limited resources. Therefore, Further research is needed to ensure the assay's reliability and robustness across diverse sample populations and varying environmental conditions. | |
| dc.identifier.uri | https://etd.aau.edu.et/handle/123456789/3527 | |
| dc.language.iso | en_US | |
| dc.publisher | Addis Ababa University | |
| dc.subject | Cas12 | |
| dc.subject | CRISPR | |
| dc.subject | crRNA | |
| dc.subject | LFD | |
| dc.subject | RPA | |
| dc.subject | Sheep and goatpox | |
| dc.title | Development of Rapid, Specific, and Field-Deployable Diagnostic Assays For Sheepox and Goatpox Viruses Using Crispr-Cas Technology | |
| dc.type | Thesis |