Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load

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art_FERREIRA FILHO_Mitochondrial_and_satellite_real_timePCR_for_detecting_T_2019.PDF (108.47 KB)
Citações na Scopus
4
Tipo de produção
article
Data de publicação
2019
DOI
Scopus
Web of Science
PubMed
Título da Revista
ISSN da Revista
Título do Volume
Editora
ACADEMIC PRESS INC ELSEVIER SCIENCE
Autores
Citação
EXPERIMENTAL PARASITOLOGY, v.200, p.13-15, 2019
Projetos de Pesquisa
Unidades Organizacionais
Fascículo
Resumo
The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 degrees C +/- 1 degrees C for satellite-DNA and 78.1 degrees C +/- 1 degrees C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 x 10(-3) parasite or 240 target copies, and for kDNA, 2 x 10(-4) parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (+/-0.002) and 0.991 (+/-0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.
Palavras-chave
Trypanosoma cruzi, Chagas disease, Molecular diagnosis, Real-time PCR, Quantitative PCR, Nuclear DNA, Satellite DNA, kDNA
URI
https://observatorio.fm.usp.br/handle/OPI/32506
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Coleções
Artigos e Materiais de Revistas Científicas - FM/MPE
Artigos e Materiais de Revistas Científicas - IMT
Artigos e Materiais de Revistas Científicas - LIM/17
Artigos e Materiais de Revistas Científicas - LIM/38
Artigos e Materiais de Revistas Científicas - LIM/48