Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis.
The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100%, 55% and 22% of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.
Instituto de Pesquisas René Rachou, Fiocruz, Belo Horizonte, MG, Brasil.
Eduardo C
EC
Ferreira
Célia M
CM
Gontijo
Israel
I
Cruz
Maria Norma
MN
Melo
Aristóbolo M
AM
Silva
D000199:Actins / Q000032:analysis / Q000235:genetics*; D000818:Animals; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D004285:Dogs; D019353:Endemic Diseases; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics*; D007896:Leishmaniasis / Q000469:parasitology / Q000662:veterinary*; D008394:Marsupialia; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012177:Retinol-Binding Proteins / Q000032:analysis / Q000235:genetics*; D012377:Rodentia; D055544:beta-Globins / Q000032:analysis / Q000235:genetics*; D000099214:Retinol-Binding Proteins, Interstitial
D000199:Actins; D017931:DNA Primers; D016054:DNA, Protozoan; D005136:Eye Proteins; D012177:Retinol-Binding Proteins; D055544:beta-Globins; D000099214:Retinol-Binding Proteins, Interstitial