Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples.

Autor: Mota TF; Laboratório de Interação Parasito-Hospedeiro e Epidemiologia (LaIPHE), Instituto Gonçalo Moniz (IGM), FIOCRUZ-BA, Rua Waldemar Falcão, Salvador, Brazil., Brodskyn CI; Laboratório de Interação Parasito-Hospedeiro e Epidemiologia (LaIPHE), Instituto Gonçalo Moniz (IGM), FIOCRUZ-BA, Rua Waldemar Falcão, Salvador, Brazil., Morello LG; Instituto Carlos Chagas (ICC), FIOCRUZ-PR, Rua Prof. Algacyr Munhoz Mader, Curitiba, Brazil.; Instituto de Biologia Molecular do Paraná (IBMP), Rua Professor Algacyr Munhoz Mader, Curitiba, Brazil., Marchini FK; Instituto Carlos Chagas (ICC), FIOCRUZ-PR, Rua Prof. Algacyr Munhoz Mader, Curitiba, Brazil.; Instituto de Biologia Molecular do Paraná (IBMP), Rua Professor Algacyr Munhoz Mader, Curitiba, Brazil., Krieger MA; Instituto Carlos Chagas (ICC), FIOCRUZ-PR, Rua Prof. Algacyr Munhoz Mader, Curitiba, Brazil., de Cássia Pontello Rampazzo R; Instituto de Biologia Molecular do Paraná (IBMP), Rua Professor Algacyr Munhoz Mader, Curitiba, Brazil., Fraga DBM; Laboratório de Interação Parasito-Hospedeiro e Epidemiologia (LaIPHE), Instituto Gonçalo Moniz (IGM), FIOCRUZ-BA, Rua Waldemar Falcão, Salvador, Brazil.; Departamento de Medicina Veterinária Preventiva e Produção Animal, Escola de Medicina Veterinária e Zootecnia, Universidade Federal da Bahia (UFBA), Salvador, Brazil.
Jazyk: angličtina
Zdroj: Medical and veterinary entomology [Med Vet Entomol] 2022 Jun; Vol. 36 (2), pp. 176-184. Date of Electronic Publication: 2022 Jan 28.
DOI: 10.1111/mve.12564
Abstrakt: 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 C q 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.
(© 2022 Royal Entomological Society.)
Databáze: MEDLINE
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