A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Abstract

Due to the overlapping distribution of

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

“…30,31 Another work showed a simplified extraction of Trypanosoma cruzi and T. rangeli DNAs in triatomine feces laid on FP. 20 In our study, we used such protocols in clinical samples from patients with ACL. The differences in the PCR positivity of the several procedures of preservation and extraction were not statistically significant.…”

“…After centrifugation at 14,000 ϫ g for 5 min at room temperature, the supernatant was used as Leishmania DNA source for the PCR reaction. 20 The extraction of DNA from EB and FB samples was performed with 100 L buffer solution (10 mM Tris-HCl and 1 mM ethylenediamine tetraacetic acid [EDTA], pH 8.0) and 100 g/mL proteinase K (final concentration), left at 56ЊC for 3 hr, and homogenized from time to time. After proteinase K inactivation by boiling for 15 min, the digested samples were centrifuged, and the supernatant was used as the Leishmania template DNA source for the PCR reaction.…”

Suitability of a rapid DNA isolation and amplification for detection ofTrypanosoma cruziinTriatoma infestansdry fecal spots collected on filter paper

“…This procedure was adopted in an attempt to homogenize the groups with respect to the number of infected insects and the total blood volume ingested considering the group as a whole. Our DNA extraction method proved to be fast and costeffective and succeed in amplifying T. cruzi DNA from day 4 post insects feeding what was more satisfactory than results obtained by others such as Machado et al (2000) who have also used a boiling procedure to extract DNA and detected T. cruzi DNA on filter paper containing triatomines feces only on day 15 after the infective meal. Conversely, in the only report in which T. cruzi DNA was detected as soon as day 2 post feeding (Russomando et al, 1996), the authors have studied digestive tract samples of insects fed on experimentally infected monkeys presenting with low parasitemia.…”

First report on the occurrence of Trypanosoma rangeli Tejera, 1920 in the state of Ceará, Brazil, in naturally infected triatomine Rhodnius nasutus Stål, 1859 (Hemiptera, Reduviidae, Triatominae)

“…Polymerase chain reaction (PCR) has been known to be more sensitive than optical microscopy for detection and specific characterization of these trypanosomes in insect vectors, mainly concerning the possibility of mixed infection (Machado et al 2000). Hence, we have used PCR for specific detection of T. rangeli and T. cruzi in samples of isolated parasites; DNA was obtained from parasite cultures using standard phenol/chloroform/ isoamyl alcohol extraction (Steindel et al 1993).…”