Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control

None of the FS-XD evaluated by qPCR amplified for X12. Nevertheless, all the EIC-FS-XD mixtures amplified for X12.We determined that X12 is useful as an EIC for XD-qPCR because we showed that the FS-XD does not contain human DNA after 30 or more days of XD incubation. This information is relevant for research on T. cruzi by XD-qPCR since it allows ruling out inhibition and false negative results due to DNA loss during the process of extraction and purification.The application of the polymerase chain reaction (PCR) has allowed the detection of Trypanosoma cruzi in several biological samples obtained from insect vectors and mammalian hosts [1-5]; however, it is a qualitative technique. By contrast, the real-time PCR (qPCR) technique allows detection and quantification of T. cruzi parasitic loads, which is particularly important in studies where the effectiveness of chemotherapy is being evaluated [6,7].Xenodiagnosis (XD) is another non-routine parasitological method, which uses biological vectors to amplify T. cruzi [8,9] and has shown good sensitivity and precocity when combined with conventional PCR [10]. The aim of this study is to evaluate the utility of a human DNA fragment as an exogenous internal control (EIC), to be used as a positive control of DNA extraction and as a qPCR inhibitor alert in studies of detection of T. cruzi by qPCR in faecal samples (FS) obtained by xenodiagnosis (FS-XD) applied in humans.All the procedures were carried out in our laboratory. We extracted 5 ml of peripheral blood by venous puncture of an individual serologically negative for Chagas disease, determined by an ELISA Chagas III kit (GrupoBios SA, Chile) and by indirect immunofluorescence IgG (IFI-IgG in-house) tests. The sample was received in 5 ml of guanidine-HCl 6 M EDTA 0.2 M, incubated at 98°C for 15 min in a double boiler and stored at 4°C. Extraction and purification were performed with an initial volume of 200 μl of blood-buffer mixture, according to the indications of th