Banca de DEFESA: ALICE MARIA SILVA DE CARVALHO

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : ALICE MARIA SILVA DE CARVALHO
DATE: 09/05/2024
TIME: 14:00
LOCAL: Videoconferência
TITLE:

"Development of Loop-Mediated Isothermal Amplification (LAMP) reaction using comparative genomics for the detection of the bacterial pathogen Erwinia psidii".

KEY WORDS:

Diagnosis, Loop-mediated isothermal amplification, dieback.

PAGES: 47
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

The bacterial disease known as "dieback," caused by Erwinia psidii, is considered a significant threat to both guava (Psidium guajava L.) and eucalyptus (Eucalyptus spp.) crops, causing losses ranging from 30 to 85%. It is one of the main limiting factors in production. Since the primary mode of disease transmission is through asymptomatic propagative plant material, sensitive, rapid, and accurate methods for detecting the pathogen in asymptomatic plants are necessary for early diagnosis. Therefore, the objective of this study is to develop a molecular detection assay using Loop-Mediated Isothermal Amplification (LAMP) for the detection of Erwinia psidii in guava and eucalyptus crops. Comparative genomic analysis using the RUCS software was conducted to identify unique genomic regions specific to E. psidii, comparing target sequences with regions of various phytopathogenic agents affecting guava and eucalyptus crops. Among the 842 unique regions identified, only the top five with the highest potential were used for primer set design. Additionally, the recA region, previously used for developing specific primers for conventional PCR and qPCR, was also selected. Seven primer sets were designed using the NEB® LAMP Primer Design Tool. Primer compatibility verification was performed using the IBSBF435 isolate, evaluating two temperatures (60 and 65 ºC) and six time intervals (30, 40, 50, 55, 60, and 75 minutes). At 65 ºC, color change occurred for four out of the seven primer sets after 50 minutes. Subsequently, an assay was conducted to examine the ratio between internal and external primers to optimize LAMP. The ratio of 8:1 (more diluted) resulted in amplification of three out of the four primer sets, while at a ratio of 4:1, all four primer sets were able to amplify. The sensitivity of the most promising primer set (Ep19) was tested with seven dilutions of DNA extracted from the isolate, ranging from 10 to 1x10-4 ng.µL-1 . The assay achieved sensitivity to detect up to 1x10-3 ng.µL-1

COMMITTEE MEMBERS:
Interna - ***.699.249-** - ANGELA MEHTA DOS REIS - UNICAMP
Externa à Instituição - Adriane Wendland Ferreira - EMBRAPA
Presidente - 3051226 - MAURICIO ROSSATO
Interna - 3117540 - THAIS RIBEIRO SANTIAGO