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Background  The PCR assays usually employed for  Leishmania  diagnosis does not simultaneously detect a constitutive gene that would certify the viability of the DNA sample. We present a multiplex PCR approach for the simultaneous diagnosis of the  Leishmania  sp. kDNA fragment and a catalytic domain segment of a conserved region of the mammalian gapdh gene.    Methodology  The proposed multiplex protocol was designed through  in silico  PCR. The annealing temperature, concentration of primer pairs, number of cycles, distinct polymerase enzymes and premix kit were defined to achieve an optimal reaction. The DNA detection sensitivity was tested with different concentrations of known  L .  tropica  DNA, and the reproducibility of the assay was confirmed using samples from 106 wild mammals that were previously subject to  Leishmania  sp. kDNA analysis through singleplex reactions.    Principal findings  The following optimal conditions were established: 95°C for 1 min followed by 30 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 1 min. The multiplex PCR system was capable of detecting 0.1 ng of  L .  tropica  diluted in 100 ng of mammalian DNA. Of 51 kDNA samples that were previously found to be positive, 45 (88.2%) were positive for both targets, two were positive only for kDNA and four were negative for both. Of 55 kDNA samples that were previously identified as negative, 38 (69.1%) were positive for gapdh whereas the other 17 were negative for both targets.    Conclusions/Significance  The proposed multiplex PCR system allows the simultaneous detection of the gapdh gene and  Leishmania  sp. kDNA in tissue samples derived from distinct wild mammal species. The amplification of the gapdh mammalian gene in the same reaction ensures the quality and viability of the DNA in the sample, eliminating the possibility of false-negative results that would impair an accurate description of the infection rates in a given population.

Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh housekeeping gene of mammal hosts