2.7. PCR analysis of the internal transcribed spacer 1 (ITS1)

Some samples were analyzed for ITS1 PCR using the primers: LITSR and L5.8S [10]. Amplification conditions were as described [12]. PCR products were digested with HaeIII enzyme, Survey of Cutaneous Leishmaniasis in Mexico: *Leishmania* Species, Clinical Expressions and Risk Factors http://dx.doi.org/10.5772/65501 157


L. am + L. mex, L. (L.) amazonensis + L. (L.) mexicana; L. b + L. mx, L. braziliensis + L. mexicana; Mx. L. mexicana, Mexican variant of L. (L.) mexicana

Table 2. Mexican isolates of Leishmania analyzed in this study.

2.4. Isolation of DNA

Table 1. Reference strains used in this study.

156 The Epidemiology and Ecology of Leishmaniasis

2.5. Polymerase chain reaction

Clinical specimens cut from the filter paper or eluted from the smear, bone marrow aspirates, skin aspirates, and tissue biopsy samples (1–2 mm) were incubated in 250 μL of cell lysis buffer for 1 h at 56°C. DNA from Leishmania cultures was prepared by centrifuging 109 parasites in the exponential phase of growth at 2000 × g for 10 min at 4°C. The DNA was extracted from the pellet using the High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer's instructions. The DNA was stored at −20°C until used.

Number Strain Code Leishmania species MHOM/BZ/82/BEL21 BEL21 L. (L.) mexicana MHOM/BZ/62/M379 M379 L. (L.) mexicana IFLA/BR/67/PH8 PH8 L. (L.) amazonensis MHOM/BR/73/M2269 M2269 L. (L.) amazonensis MHOM/PE/84/LC53 LC53 L. (V.) braziliensis MHOM/BR/84/LTB300 LTB300 L. (V.) braziliensis MHOM/BR/75/M2903 M2903 L. (V.) braziliensis MHOM/BR/75/M2904 M2904 L. (V.) braziliensis MHOM/BR/75/M4147 M4147 L. (V.) guyanensis MHOM/PE/84/LC26 LC26 L. (V.) peruviana MHOM/CR/87/NEL3 NEL3 L. (V.) panamensis MHOM/PA/72/LS94 LS94 L. (V.) panamensis MHOM/IN/80/DD8 DD8 L. (L.) donovani MHOM/BR/74/PP75 PP75 L. (L.) infantum/chagasi

PCR analysis of kDNA for subgenus Leishmania was carried out by using the AJS1 and DeB8 primers [13]. PCR of the L. mexicana complex was carried out using the M1 and M2 primers [14] and the LMO1 and LMO2 primers specific for minicircles of Mexican L. (L.) mexicana strains [15]). PCR of the L. braziliensis complex was done with the B1 and B2 primers [8]. PCR for L. donovani complex was done with the D1 and D2 primers [16]. PCR amplification condi-

PCR species specific for nuclear DNA from variants of L. (V.) braziliensis was carried out by using the primers 3J1 and 3J2. Amplification conditions were as described elsewhere [18].

Some samples were analyzed for ITS1 PCR using the primers: LITSR and L5.8S [10]. Amplification conditions were as described [12]. PCR products were digested with HaeIII enzyme,

tions were performed as described previously [8, 13, 14, 16, 17].

2.6. PCR analysis of genomic DNA of L. (V.) braziliensis

2.7. PCR analysis of the internal transcribed spacer 1 (ITS1)

according to the manufacturer's instructions. The amplicons and restriction products were analyzed as described elsewhere [12].
