Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis

Conventional nested PCR is a very sensitive and specific method for the diagnosis of visceral leishmaniasis. However, this type of PCR is notorious for contamination problems related to the processing of the product between the first and the second PCR steps. In order to have a PCR method that is just as efficient but without the risk of contamination, we attempted the optimization of a single-tube nested PCR (STNPCR) method. During the first and the second PCR steps, we used the small subunit of ribosomal RNA (ssu rRNA) and the ribosomal internal transcribed spacer (ITS) as targets, respectively. The performances of STNPCR and nested PCR in detecting the DNA of Leishmania chagasi were compared. In the case of STNPCR, the inner primers were immobilized on the interior of the tube cap by means of adsorption microtubes and then were solubilized before the second reaction. This procedure eliminated the need to open the microtube, which could have led to false-positive results through cross-contamination. The detection limit for the purified L. chagasi DNA was 1 fg by using nested PCR and 10 fg by using STNPCR. We also tested the specificity of the system against other parasites, and observed that Trypanosoma cruzi DNA was amplified with a detection limit of up to 1 pg. This study not only presents a promising tool for the diagnosis of visceral leishmaniasis, but also provides a new tool for the diagnosis of Chagas disease, either in mono-infection by T. cruzi or in co-infection with Leishmania spp.