Author | Mota, Tiago Feitosa | |
Author | Brodskyn, Claudia Ida | |
Author | Morello, Luis Gustavo | |
Author | Marchini, Fabricio Klerynton | |
Author | Krieger, Marco Aurelio | |
Author | Rampazzo, Rita de Cássia Pontello | |
Author | Fraga, Deborah Bittencourt Mothé | |
Access date | 2022-02-25T15:16:00Z | |
Available date | 2022-02-25T15:16:00Z | |
Document date | 2022 | |
Citation | MOTA, Tiago Feitosa et al. Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples. Medical and Veterinary Entomology, p. 1-9, 2022. | pt_BR |
ISSN | 1365-2915 | pt_BR |
URI | https://www.arca.fiocruz.br/handle/icict/51494 | |
Language | por | pt_BR |
Publisher | Wiley | pt_BR |
Rights | open access | pt_BR |
Subject in Portuguese | multiplex qPCR | pt_BR |
Subject in Portuguese | phlebotomine | pt_BR |
Title | Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples | pt_BR |
Type | Article | pt_BR |
DOI | 10.1111/mve.12564 | |
Abstract | The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer–probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is,
individual midguts. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. | pt_BR |
Affilliation | Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil / Universidade Federal da Bahia. Escola de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Produção Animal. Salvador, BA, Brasil. | pt_BR |
Subject | Genes Insect | pt_BR |
Subject | Multiplex Polymerase Chain Reaction | pt_BR |
Subject | Leishmaniasis, Visceral | pt_BR |
Subject in Spanish | Genes de Insecto | pt_BR |
Subject in Spanish | Reacción en Cadena de la Polimerasa Multiplex | pt_BR |
Subject in Spanish | Leishmaniasis Visceral | pt_BR |
Subject in French | Gènes d'insecte | pt_BR |
Subject in French | Réaction de polymérisation en chaine multiplex | pt_BR |
Subject in French | Leishmaniose viscérale | pt_BR |
DeCS | Genes de Insetos | pt_BR |
DeCS | Reação em Cadeia da Polimerase Multiplex | pt_BR |
DeCS | Phlebotominae | pt_BR |
DeCS | Leishmaniose Visceral | pt_BR |