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Banca de QUALIFICAÇÃO: LETICIA MIKARDYA LIMA SALES

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
STUDENT : LETICIA MIKARDYA LIMA SALES
DATE: 06/08/2024
TIME: 10:00
LOCAL: Link de acesso para videoconferência: https://meet.google.com/kxz-jucr-mdt?authuser=0
TITLE: ENTOMOLOGICAL SURVEILLANCE OF CHAGAS DISEASE VECTORS IN RIO GRANDE DO NORTE, BRAZIL

KEY WORDS:

Trypanosoma cruzi. Primers 18S. COX II. NADH 1. NADH 5.

PAGES: 72
BIG AREA: Ciências da Saúde
AREA: Farmácia
SUMMARY:

Chagas disease (CD) is a parasitic infection caused by the flagellated protozoan Trypanosoma

cruzi, which presents variable pathological and symptomatic characteristics. The diagnosis of

this infection is based mainly on serological methods, however, inconclusive or false positive

results are recurrent, mainly due to cross-reactions with other trypanosomatids. Thus, advances

in molecular biology are responsible for significant improvements in the diagnosis of infection.

The objective of this study was the identification of T. cruzi by DNA amplification with new

primers in the Polymerase Chain Reaction (PCR). The design of the primers was based on the

DNA sequences of T. cruzi reference strains, clones and isolates, deposited in NCBI GenBank

and listed by Zingales et al. (2009) for the kDNA genes: 9S, 18S, Cytochrome B, Cytochrome

Oxidase subunit II (COX II), NADH dehydrogenase subunit 1 (NADH 1) and NADH

dehydrogenase subunit 5 (NADH 5). The primers were designed in the Primer Blast NCBI

software. After manual checking and in silico tests, the primers that showed the lowest coverage

rate of the target gene were excluded from the study, leaving the most promising primers,

designed for the 18S gene (5' – CAAGCGGCTGGGTGGTTATT – 3' and 3' –

CACGGATTTCCCACAAAGGC – 5') , COX II (5' – TGTTATCCATTCATTTACGTTAGC

–

3' and 3'

–

CATAACTCGCTGCATTGC

–

5'), NADH 1 (5'

–

AAGTCCAGCAACCAATTCACTT – 3' and 3' – CGTTACTCTGTGATGGCTTGA – 5') and

NADH 5 (5'

–

AGAGTACACAGTTTGGGTTG

–

3' and 3'

–

CCACATACAACTAACGTTGC – 5'). Next, modifications to the PCR conditions were tested

for the four chosen primer pairs and annealing temperatures of 60ºC (18S), 48ºC (COX II),

57ºC (NADH 1) and 55ºC (NADH 5) were defined. From the analytical sensitivity test of the

concentration of parasites in the blood and the concentration of parasite DNA in the sample, it

was possible to determine that the primers designed for 18S were more sensitive and detected

the parasite at concentrations of 0.1fg in TcI (Col 1.7); from 1000fg to 1fg in TcII (Y) and

1000fg to 10fg in TcIII (CBS56) and from 0.5p/mL to 1000p/mL in DTUs TcII and TcIII. The

primers used for amplification of the NADH 1 gene presented a fragment of approximately

71bp, which appeared only at a concentration of 1000fg of the Col. 1.7 strain. These primers

were unable to detect the DTUs TcII and TcIII at any concentration. The NADH 5 primers

showed a band pattern of 100bp of TcI DNA, at a concentration of 1000fg, and TcII and TcIII,

at concentrations of 10fg to 1000fg. The primers were able to detect T. cruzi DNA adequately

in small concentrations and amplified non-specific bands in human blood samples.

COMMITTEE MEMBERS:
Externo ao Programa - 1046091 - JOAO FIRMINO RODRIGUES NETO - nullPresidente - 1055045 - MARCELA ABBOTT GALVAO URURAHY
Externa ao Programa - 1715271 - RENATA ANTONACI GAMA - null

Notícia cadastrada em: 24/07/2024 14:00