Addressing dengue virus detection challenges with a low-cost and portable RT-LAMP-based assay
Dengue virus (DENV) causes the greatest human disease burden of any arthropod-borne virus, with about 390 million infections per year and 100 million symptomatic cases. As no vaccine or specific antiviral treatment is available, early recognition of the infection is paramount, since correct diagnosi...
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| Tipo de recurso: | tesis de maestría |
| Estado: | Versión aceptada para publicación |
| Fecha de publicación: | 2024 |
| País: | Colombia |
| Institución: | Universidad de los Andes |
| Repositorio: | Séneca: repositorio Uniandes |
| Idioma: | inglés |
| OAI Identifier: | oai:repositorio.uniandes.edu.co:1992/74526 |
| Acceso en línea: | https://hdl.handle.net/1992/74526 |
| Access Level: | acceso abierto |
| Palabra clave: | RT-LAMP Dengue virus Diagnostics Homemade enzymes Cell free protein synthesis Aptaswitches Biología |
| Sumario: | Dengue virus (DENV) causes the greatest human disease burden of any arthropod-borne virus, with about 390 million infections per year and 100 million symptomatic cases. As no vaccine or specific antiviral treatment is available, early recognition of the infection is paramount, since correct diagnosis helps to guide supportive clinical care, resulting in timely and appropriate clinical interventions that prevent severe morbidity and mortality. Loop-mediated isothermal amplification (LAMP) is a popular amplification method because it does not require a thermocycler and has a reduced cost and complexity compared to PCR-based assays. LAMP-based non-specific amplification can be circumvented by additional readout methods such as the recently reported programmable aptamer-based RNA switches, or aptaswitches, that activate rapidly, provide a strong fluorescent readout, and exhibit great sensitivity. In low-resourced areas where access to health care is limited, the high cost of imported, commercial enzymes can pose significant challenges to deploying and implementing LAMP-based detection methods. Here, we developed a low-cost-portable RT-LAMP-based assay capable of detecting DENV in vitro using two homemade enzymes produced via CFPS: Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and Bst large fragment (BstLF) DNA polymerase. Enzymes were successfully produced using cell-free protein synthesis and purified in just two days. The homemade RT-LAMP presented a limit of detection (LoD) of 10 copies/μL for DENV-1 and a LoD between 100 and 10 copies/μL for DENV-2, and the RT-LAMP/aptaswitch reaction presented a LoD of 5 copies/μL, meaning it is sensitive enough to detect DENV in patients with an active infection. A lyophilization assay of the homemade RT-LAMP reactions was also successful, the results show that the enzymes in the 10% trehalose buffer can be successfully freeze-dried and maintain their activity after rehydration. The resulting diagnostic test for DENV provides comparable sensitivity to commercial RT-LAMP methods and has point of care (PoC) application potential in a country like Colombia where DENV is hyperendemic and alternative laboratory confirmation tests are urgently needed to confirm infection. Taken together, the work presented provides an opportunity for the development of “made in Colombia” diagnostics, with clinically relevant sensitivity, that have the potential to improve the accessibility to the surveillance and detection of this prevalent disease, reducing its morbidity and mortality. |
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