SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases

• Main Authors: Pita-Pereira Daniela, Lins Rachel, Oliveira Marcia P, Lima Rosimar B, Pereira Bernardo AS, Moreira Otacilio C, Brazil Reginaldo P, Britto Constança
• Format: Article
• Language: English
• Published: BMC 2012-01-01
• Series: Parasites & Vectors
• Subjects: SYBR Green Real-time PCR; Leishmaniases; kinetoplast DNA; thermal dissociation curves; molecular diagnosis; Brazil
• Online Access: http://www.parasitesandvectors.com/content/5/1/15

<p>Abstract</p> <p>Background</p> <p>Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between <it>Leishmania </it>isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect <it>Leishmania </it>infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.</p> <p>Results</p> <p>A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between <it>Leishmania </it>subgenera. A panel of reference strains representing subgenera <it>Leishmania </it>and <it>Viannia </it>was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95°C ± 0.01 and 77.36°C ± 0.02 for <it>Leishmania </it>and <it>Viannia</it>, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together <it>L</it>. (<it>V</it>.) <it>braziliensis </it>and <it>L</it>. (<it>V</it>.) <it>shawii </it>with a bootstrap value of 100%; while for <it>L</it>. (<it>L</it>.) <it>infantum </it>and <it>L</it>. (<it>L</it>.) <it>amazonensis</it>, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus <it>Viannia </it>amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus <it>Leishmania </it>(average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected <it>Lutzomyia </it>spp. captured in two Brazilian localities.</p> <p>Conclusions</p> <p>For all tested samples, a characteristic amplicon melting profile was evidenced for each <it>Leishmania </it>subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.</p>