Serial quantitative PCR assay for detection, species-discrimination and quantification of Leishmania spp. in human samples

A method for determining the presence and species of a Leishmania in a sample, and distinguishing the Leishmania species so determined from other Leishmania species of interest present in the sample, the method comprising:

• (a) contacting the sample with the forward-and-reverse primer pairs GGGTAGGGGCGTTCTGC (SEQ ID NO:1) and TACACCAACCCCCAGTTTGC (SEQ ID NO:
• 2) (kDNA 1 minicircle forward and reverse primers, respectively);
• TGCTATAAAATCGTACCACCCGACA (SEQ ID NO:
• 101) and GAACGGGGTTTCTGTATGCCATTT (SEQ ID NO:
• 102) (L. (Y) braziliensis kDNA 3 forward and reverse primers, respectively);
• AGAGCGTGCCTTGGATTGTG (SEQ ID NO:
• 49) and CGCTGCGTTGATTGCGTTG (SEQ ID NO:
• 50) (MSP Associated Gene I (MAG I) forward and reverse primers, respectively);
• and GGAGAAACTCACGGCACAGG (SEQ ID NO:
• 67) and GCGCCTCGTAGGTCACAGTT (SEQ ID NO:
• 68) (SLACS forward and reverse primers, respectively);
• thereby forming Leishmania-primer pair complexes;
• (b) exposing the Leishmania-primer pair complexes of (a) to a thermo-stable polymerase and deoxyribonucleoside triphosphates to produce (1) double-stranded DNAs containing the primer sequences and (2) pyrophosphates;
• (c) performing temperature cycles to permit further production of additional double-stranded DNAs containing the primer sequences and additional pyrophosphates;
• (d) determining the melting temperature of the double-stranded DNA product of (c), a characteristic melting temperature being indicative of particular Leishmania species so detected by the forward-and-reverse primer pairs of (a);
• and
• (e) comparing the melting temperature of (d) to a characteristic melting temperature for particular Leishmania species so detected by the forward-and-reverse primer pairs of (a);
• thereby determining the presence and the species of the Leishmania in the sample, wherein the Leishmania species of interest comprises any of Leishmania tropica, Leishmania chagasi/Leishmania infantum, Leishmania donovani, Leishmania major, Leishmania braziliensis, Leishmania guyanensis/Leishmania panamensis, Leishmania mexicana, and Leishmania amazonensis.