LIMITATION OF PRIMERS USED IN PCR FOR THE CHARACTERIZATION OF LEISHMANIA INFANTUM.
J Parasitol
; 109(5): 445-449, 2023 10 01.
Article
em En
| MEDLINE
| ID: mdl-37668295
Conventional PCR provides Leishmania species characterization with even a small amount of biological material. Species-specific primers have been a widely used alternative; however, nonspecific amplifications are a reality, interfering with PCR efficiency. In endemic areas with multiple etiological agents for leishmaniasis, there is a requirement for higher specificity of primers. This study evaluates 3 pairs of primers described for the identification and characterization of Leishmania infantum. Primers RV1/RV2, LEISH1/LEISH2, and FLC2/RLC2 were used with the DNA of L. infantum, Leishmania amazonensis, and Leishmania braziliensis. An initial temperature curve was performed (52-62 C) to determine the optimal annealing temperature, followed by a dilution curve of Leishmania DNA (500 pg/µl, 50 pg/µl, 5 pg/µl, 500 fg/µl, 50 fg/µl, 5 fg/µl, and 0.5 fg/µl) to be used for analytical sensitivity. RV1/RV2 PCR amplified L. infantum and L. amazonensis at all analyzed temperatures; LEISH1/LEISH2 PCR amplified all 3 species of Leishmania, although at some temperatures L. infantum was specifically amplified, and, finally, FLC2/RLC2 PCR amplified only L. infantum at all temperatures analyzed. In terms of sensitivity, RV1/RV2 PCR detected 1 fg of L. infantum DNA and 100 pg of L. amazonensis DNA; LEISH1/LEISH2 PCR detected 1 fg of L. infantum DNA, 100 fg of L. amazonensis DNA, and 10 fg of L. braziliensis DNA; and FLC2/RLC2 PCR detected 10 fg of L. infantum DNA. Thus, PCR with FLC2/RLC2 primers is best suited for the molecular characterization of L. infantum, especially in areas where there is an incidence of more than 1 Leishmania species, such as South America.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Leishmania mexicana
/
Leishmania infantum
País/Região como assunto:
America do sul
Idioma:
En
Revista:
J Parasitol
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Estados Unidos