Banca de DEFESA: LETICIA MIKARDYA LIMA SALES

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : LETICIA MIKARDYA LIMA SALES
DATE: 28/01/2025
TIME: 09:00
LOCAL: VIDEOCONFERÊNCIA
TITLE:

Identification of Trypanosoma cruzi by DNA Amplification Using New Primers in the Polymerase Chain Reaction (PCR)

KEY WORDS:

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

PAGES: 96
BIG AREA: Ciências da Saúde
AREA: Farmácia
SUMMARY:

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

Trypanosoma cruzi. The diagnosis of the chronic phase of Chagas disease is

performed mainly by serological tests, which detect specific anti-T. cruzi antibodies.

However, inconclusive or false-positive results are recurrent, mainly due to cross

reactions with other trypanosomatids. Thus, advances in molecular biology are

responsible for significantly improving the diagnosis of the infection. The objective of

this study was to identify T. cruzi from different DTUs by DNA amplification with new

primers in the Polymerase Chain Reaction (PCR). Primer design was based on DNA

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

GenBank 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). Primers were designed using NCBI Primer Blast software. After

manual checking and in silico testing, the most promising primers designed for the 18S

gene (5’

–

CAAGCGGCTGGGTGGTTATT

–

3’ and 3’

–

CACGGATTTCCCACAAAGGC – 5’) produced a 104bp fragment, COX II (5’ –

TGTTATCCATTCATTTACGTTAGC – 3’ and 3’ – CATAACTCGCTGCATTGC – 5’)

produced a 122bp fragment, NADH 1 (5’ – AAGTCCAGCAACCAATTCACTT – 3’ and

3’ – CGTTACTCTGTGATGGCTTGA – 5’) formed a 71bp band and NADH 5 (5’ –

AGAGTACACAGTTTGGGTTG – 3’ and 3’ – CCACATACAACTAACGTTGC – 5’),

which produced a 100bp band were selected for the study. Initially, the PCR conditions

were tested for the four chosen primer pairs and the annealing temperatures were

defined as 60ºC (18S), 48ºC (COX II), 57ºC (NADH 1) and 55ºC (NADH 5). Then, the

analytical sensitivity test was performed with different DTUs, DNA concentrations and

parasite quantity. The primers designed for 18S detected the parasite at concentrations

of 1000fg to 0.1fg in TcI (Col 1.7), from 1000fg to 1fg in TcII (Y) and 1000fg to 10fg in

TcIII (CBS56). The analytical sensitivity of the parasite concentration in the blood

sample revealed that the 18S primers were able to detect T. cruzi DNA at

concentrations of 1000p/mL in TcI and TcII samples; and from 10pmL to 1000p/mL in

TcIII. The COXII PCR did not achieve adequate standardization. The primers used for

the NADH 1 and NADH 5 PCR were not able to detect all the DTUs tested. The

identification of T. cruzi was not very efficient in human blood samples, from

domesticated animals such as Canis familiaris, Ovis aries and Capra hircus and the

parasite DNA was identified in all T. cruzi samples from cultures of intestinal contents

of triatomines.

COMMITTEE MEMBERS:
Presidente - 1218940 - ANTONIA CLAUDIA JACOME DA CAMARA
Externo ao Programa - 1046091 - JOAO FIRMINO RODRIGUES NETO - UFRNExterno à Instituição - ELIANE LAGES SILVA - UFTM