Desarrollo y optimización de la técnica de amplificación isotérmica mediada por loops (LAMP) para la elaboración de un kit diagnóstico de leptospirosis animal : Lepto-LAMP
Leptospirosis is considered one of the most widespread zoonotic diseases in the world. In Argentina, it is endemic, with outbreaks occurring mainly during periods of abundant precipitation. Leptospira spp. is the second most important abortifacient pathogen in livestock production, responsible for t...
Guardado en:
| Autor principal: | |
|---|---|
| Otros Autores: | |
| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
| Publicado: |
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2024
|
| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7984 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7984.dir/7984.PDF |
| Aporte de: |
| Sumario: | Leptospirosis is considered one of the most widespread zoonotic diseases in the world. In Argentina, it is endemic, with outbreaks occurring mainly during periods of abundant precipitation. Leptospira spp. is the second most important abortifacient pathogen in livestock production, responsible for the loss of millions of dollars in the beef and dairy industry in the Pampas region. In addition to the economic impact on animal industries, this zoonosis represents a risk to humans who are in constant contact with infected animals, whether domestic or wild. For these reasons, it is crucial to have diagnostic tools that allow simple and effective detection of the agent, in order to implement necessary measures to prevent spread of the pathogen and mitigate economic and health consequences.\nThe aim of this PhD thesis was to develop a method for detecting leptospiral DNA based on loop-mediated isothermal amplification (LAMP) technique, designed for application in low-complexity laboratories, using national supplies and easy handling: Lepto-LAMP.\nInitially, two DNA extraction methods were evaluated, with Chelex®100 resin chosen for serum and animal urine samples due to its high yield, despite not requiring an additional purification step. On the other hand, the PURO Genomic DNA® kit was selected for samples of animal organs, as it allowed for purer DNA extraction than the resin.\nDuring the design of the LAMP reaction for the detection of leptospiral DNA, five possible indicators for direct visual result reading were tested: pH indicators, malachite green, SYBR Safe®, hydroxynaphthol blue, and calcein; in addition to evaluating different reaction times. After optimization, Lepto-LAMP incorporated calcein as an indicator due to its higher analytical sensitivity (10 pg of DNA per reaction, or 0.1; 1 and 10 leptospires/mL in bovine serum, urine, and kidney macerate, respectively). Lepto-LAMP specifically detected DNA from pathogenic and intermediate strains of Leptospira spp.\nLepto-LAMP was compared with the reference technique PCR lipL32 for the detection of leptospiral DNA in animal clinical samples. In a trial using hamsters (Mesocricetus auratus) as a model of animal leptospirosis, Lepto-LAMP allowed for the detection of a greater number of positive samples in infected animals. Both molecular diagnostic techniques showed substantial agreement according to Cohen's Kappa coefficient. Employing clinical samples (serum, urine, and organs) from domestic and wild animals, Lepto-LAMP demonstrated high diagnostic sensitivity (100%, 95% CI: 93.6?100%) and high diagnostic specificity (96.6 %, 95 % CI: 95.2-97.7 %). Lepto-LAMP had a lower implementation cost compared to the endpoint PCR lipL32, making it more accessible for low-complexity laboratories.\nThe use of Lepto-LAMP for the diagnosis of animal leptospirosis has great potential to complement the microscopic agglutination test, the gold standard serological technique for leptospirosis, as it can be implemented in most non-specialized laboratories and provides a short time to obtain results (between 75-90 minutes from DNA extraction with Chelex®100 to final result reading). |
|---|