Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3′ untranslated region of the heat shock protein 70 (type I) gene

PCR-based methods to amplify the 3′ untranslated region (3′-UTR) of the heat shock protein 70 (type I) gene ( HSP70-I ) have previously been used for typing of Leishmania but not with Leishmania ( Mundinia ) martiniquensis and L . ( Mundinia ) orientalis , newly identified human pathogens. Here, the 3′-UTRs of HSP70-I of L . martiniquensis , L . orientalis , and 10 other species were sequenced and analyzed. PCR-Restriction Fragment Length Polymorphism (RFLP) analysis targeting the 3′-UTR of HSP70-I was developed. Also, the detection limit of HSP70-I -3′-UTR PCR methods was compared with two other commonly used targets: the 18S small subunit ribosomal RNA (SSU-rRNA) gene and the internal transcribed spacer 1 region of the rRNA (ITS1-rRNA) gene. Results showed that HSP70-I -3′-UTR PCR methods could be used to identify and differentiate between L . martiniquensis (480–2 bp) and L . orientalis (674 bp) and distinguished them from parasites of the subgenus Viannia and of the subgenus Leishmania . PCR-RFLP patterns of the 3′-UTR of HSP70-I fragments digested with Bsu RI restriction enzyme successfully differentiated L . martiniquensis , L . orientalis , L . braziliensis , L . guyanensis = L . panamensis , L . mexicana = L . aethiopica = L . tropica , L . amazonensis , L . major , and L . donovani = L . infantum . For the detection limit, the HSP70-I -3′-UTR PCR method could detect the DNA of L . martiniquensis and L . orientalis at the same concentration, 1 pg/μL, at a similar level to the SSU-rRNA PCR. The PCR that amplified ITS1-rRNA was more sensitive (0.01 pg/μL) than that of the HSP70-I -3′-UTR PCR. However, the sizes of both SSU-rRNA and ITS1-rRNA PCR amplicons could not differentiate between L . martiniquensis and L . orientalis . This is the first report of using HSP70-I -3′-UTR PCR based methods to identify the parasites causing leishmaniasis in Thailand. Also, the Bsu RI-PCR-RFLP method can be used for differentiating some species within other subgenera.