Serial quantitative PCR assay for detection, species-discrimination and quantification of

1. A method for determining the presence of Leishmania species selected from the group consisting of: Leishmania tropica, Leishmania chagasi, Leishmania infantum, Leishmania donovani, Leishmania major, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania mexicana , and Leishmania amazonensis in a sample, and distinguishing the Leishmania species so determined from other Leishmania species present in the sample, the method comprising the following steps:

(a) forming Leishmania -primer pair complexes by contacting the sample with a nucleic acid primer pair selected from the group consisting of:

(SEQ ID NO: 1)

GGGTAGGGGCGTTCTGC

and

(SEQ ID NO: 2)

TACACCAACCCCCAGTTTGC

(kDNA 1 minicircle forward and reverse primers,

respectively);

(SEQ ID NO: 97)

AACTTTTCTGGTCCTCCGGGTAG

and

(SEQ ID NO: 98)

ACCCCCAGTTTCCCGCC

(kDNA 2 forward and reverse primers, respectively);

(SEQ ID NO: 3)

GGGTAGGGGCGTTCTGC

and

(SEQ ID NO: 4)

CCCGGCCTATTTTACACCAACC

(kDNA 3 minicircle forward and reverse primers,

respectively);

(SEQ ID NO: 5)

GGGTGCAGAAATCCCGTTCA

and

(SEQ ID NO: 6)

CCCGGCCCTATTTTACACCA

(kDNA 4 minicircle forward and reverse primers,

respectively);

(SEQ ID NO: 99)

CTTTTCTGGTCCTCCGGGTAGG

and

(SEQ ID NO: 100)

CCACCCGGCCCTATTTTACACCAA

(kDNA 5 minicircle forward and reverse primers,

respectively);

(SEQ ID NO: 7)

AATGGGTGCAGAAATCCCGTTC

and

(SEQ ID NO: 8)

CCACCACCCGGCCCTATTTTAC

(kDNA 7 minicircle forward and reverse primers,

respectively);

(SEQ ID NO: 9)

GGTCCCGGCCCAAACTTTTC

and

(SEQ ID NO: 10)

CCGGGGTTTCGCACTCATTT

( L . ( L .)  amazonensis  kDNA 1 forward and reverse

primers, respectively);

(SEQ ID NO: 11)

GGTAGGGGCGTTCTGCGAAT

and

(SEQ ID NO: 12)

CCCGGCCTATTTTACACCAACC

( L . ( L .)  amazonensis  kDNA 2 forward and reverse

primers, respectively);

(SEQ ID NO: 13)

GGGTAGGGGCGTTCTGC

and

(SEQ ID NO: 14)

TACACCAACCCCCAGTTTGC

( L . ( L .)  amazonensis  kDNA 3 forward and reverse

primers, respectively);

(SEQ ID NO: 15)

TGAGTGCAGAAACCCCGTTCATA

and

(SEQ ID NO: 16)

ACACCAACCCCCAGTTGTGA

( L . ( L .)  amazonensis  kDNA 4 forward and reverse

primers, respectively);

(SEQ ID NO: 17)

AATTTCGCAGAACGCCCCTAC

and

(SEQ ID NO: 18)

GTACTCCCCGACATGCCTCTG

( L  ( V .)  braziliensis  kDNA 1 forward and reverse

primers, respectively);

and

(SEQ ID NO: 101)

TGCTATAAAATCGTACCACCCGACA

and

(SEQ ID NO: 102)

GAACGGGGTTTCTGTATGCCATTT

( L  ( V .)  braziliensis  kDNA 3 forward and reverse

primers, respectively);

(b) producing pyrophosphates and double-stranded DNAs containing the primer sequences by exposing the Leishmania -primer pair complexes to a thermo-stable polymerase and deoxyribonucleoside triphosphates;

(c) producing additional pyrophosphates and double-stranded DNAs containing the primer sequences by performing temperature cycles;

(d) determining the melting temperature of the double-stranded DNA produced;

(e) comparing the determined melting temperature to a melting temperature that is characteristic of a particular Leishmania species detected by the selected nucleic acid primer pair;

(f) determining whether the particular Leishmania species is present in the sample based on the comparison; and

(g) determining which of the Leishmania species of the group are present in the sample by repeating the steps in (a) to (f) using a different nucleic acid primer pair selected from the group consisting of:

(SEQ ID NO: 103)

TCCGCAGGAGACTTCGTATG

and

(SEQ ID NO: 104)

CACGACTATCCACCCCATCC

( L  ( L .)  infantum  Minicircle 1 forward and reverse

primers, respectively);

(SEQ ID NO: 19)

ACGGGGTTTCTGCACCCATT

and

(SEQ ID NO: 20)

GTAGGGGCGTTCTGCGAAAA

( L . ( L .)  major  Minicircle 1 forward and reverse

primers, respectively);

(SEQ ID NO: 21)

AATGCGAGTGTTGCCCTTTTG

and

(SEQ ID NO: 22)

GCCGAACAACGCCATATTAACC

( L . ( L .)  mexicana  Minicircle 1 forward and reverse

primers, respectively);

(SEQ ID NO: 23)

GGGGGTTGGTGTAAAATAGGG

and

(SEQ ID NO: 24)

ACCACCAGCAGAAGGTCAAAG

( L . ( L .)  tropica  Minicircle 1 forward and reverse

primers, respectively);

(SEQ ID NO: 25)

GCGGTGGCTGGTTTTAGATG

and

(SEQ ID NO: 26)

TCCAATGAAGCCAAGCCAGT

( L . ( L .)  donovani  Minicircle 1 forward and reverse

primers, respectively);

(SEQ ID NO: 27)

ATTTTAGTATGAGTGGTAGGTTTTGTT

and

(SEQ ID NO: 28)

CAATAACTGGGACGGTTGCT

(Cytochrome B 1 forward and reverse primers,

respectively);

(SEQ ID NO: 29)

GCGGAGAGGAAAGAAAAGGCTTA

and

(SEQ ID NO: 30)

AAAAGTCATGCTAAACACACACCACA

( L . ( L .)  amazonensis  Cytochrome B1 forward and

reverse primers, respectively);

(SEQ ID NO: 31)

CAGGTTGCTTACTACGTGTTTATGGTG

and

(SEQ ID NO: 32)

TCGTATTACAAACCCTAAATCAAAATCTCA

( L . ( L .)  tropica  Cytochrome B 1 forward and

reverse primers, respectively);

(SEQ ID NO: 33)

TCAGGTTGCTTACTACGTGTTTATGGTG

and

(SEQ ID NO: 34)

TGCTAAACAAACACCACATATGATCTGC

( L . ( L .)  tropica  Cytochrome B 2 forward and reverse

primers, respectively);

(SEQ ID NO: 35)

TGACACACATATTTTAGTGTGGGTGGTAGG

and

(SEQ ID NO: 36)

TCCCCAATAAGACATCATTGTACATGGTAA

( L . ( L .)  tropica  Cytochrome B 3 forward and reverse

primers, respectively);

(SEQ ID NO: 37)

CACATATTTTAGTGTGGGTGGTAGGTTTTG

and

(SEQ ID NO: 38)

TCCCCAATAAGACATCATTGTACATGGTAA

( L . ( L .)  tropica  Cytochrome B 4 forward and reverse

primers, respectively);

(SEQ ID NO: 39)

GCTTGGTTGGATTATTTTTGCTG

and

(SEQ ID NO: 40)

AACAACATTTTAACTCTTGTAGGATTCG

(Maxicircle 1 forward and reverse primers,

respectively);

(SEQ ID NO: 41)

GAGGTGTTTGCCCGCATC

and

(SEQ ID NO: 42)

CTCGCCCATGTCGTCG

(Alpha-tubulin 1 forward and reverse primers,

respectively);

(SEQ ID NO: 105)

TGTCGCTTGCAGACCAGATG

and

(SEQ ID NO: 106)

GCATCGCAGGTGTGAGCA

(DNA polymerase 1 forward and reverse primers,

respectively);

(SEQ ID NO: 43)

AGGAGGATGGCAAGCGGAAG

and

(SEQ ID NO: 44)

GCGACGGGTACAGGGAGTTG

(DNA polymerase 2 forward and reverse primers,

respectively);

(SEQ ID NO: 45)

CGAAACTTCCGGAACCTGTCTT

and

(SEQ ID NO: 46)

CACCACACGCACGCACAC

(Mini-exon 1 forward and reverse primers,

respectively);

(SEQ ID NO: 47)

GTGTGGTGGCGGGTGTATGT

and

(SEQ ID NO: 48)

GCCCAGGTCGCTGTGAGG

(Mini-exon 2 forward and reverse primers,

respectively);

(SEQ ID NO: 49)

AGAGCGTGCCTTGGATTGTG

and

(SEQ ID NO: 50)

CGCTGCGTTGATTGCGTTG

(MSP Associated Gene 1 (MAG 1) forward and reverse

primers, respectively);

(SEQ ID NO: 51)

AGTTTTGGTTGGCGCTCCTG

and

(SEQ ID NO: 52)

CCCACTCGCTTTCCTTGGTC

(MSP Associated Gene 2 (MAG 2) forward and reverse

primers, respectively);

(SEQ ID NO: 53)

CGACCCTGTCACCACCACAG

and

(SEQ ID NO: 54)

GAGGCCACCCTATCGCTGAC

(SIDER repeat 1 forward and reverse primers,

respectively);

(SEQ ID NO: 55)

TCGTTGAGGGAGGAGGTGTTTC

and

(SEQ ID NO: 56)

TCGGCTTTGAGGTTGGCTTC

( L . ( V .)  braziliensis  DNA polymerase 1 forward and

reverse primers, respectively);

(SEQ ID NO: 57)

ACGTCGCCAACTGCTTCACC

and

(SEQ ID NO: 58)

GTGTTCGCACCGCCTTGAC

( L . ( V .)  braziliensis  DNA polymerase 2 forward and

reverse primers, respectively);

(SEQ ID NO: 59)

GTCGTTGTCCGTGTCGCTGT

and

(SEQ ID NO: 60)

CGCTGTGTGTGTCCGTGTGT

( L . ( L .)  major  MSP associated gene 1 (L. major

MAG 1) forward and reverse primers, respectively);

(SEQ ID NO: 61)

GACGACGACGAGGAGGATGG

and

(SEQ ID NO: 62)

GCGACGGGTACAGGGAGTTG

( L  ( L .)  amazonensis  DNA polymerase 1 forward and

reverse primers, respectively);

(SEQ ID NO: 107)

CCAGATGCCGACCAAAGC

and

(SEQ ID NO: 108)

CGCGCACGTGATGGATAAC

(GPI forward and reverse primers, respectively);

(SEQ ID NO: 63)

GAAGGTGCAGTCCCTCGTGT

and

(SEQ ID NO: 64)

CCTCCGTCTGCTTGCTCTTG

(HSP70-1 forward and reverse primers,

respectively);

(SEQ ID NO: 65)

TCGAGATCGACGCGTTGTT

and

(SEQ ID NO: 66)

CCGCACAGCTCCTCGAA

(HSP70-4 forward and reverse primers,

respectively);

and

(SEQ ID NO: 67)

GGAGAAACTCACGGCACAGG

and

(SEQ ID NO: 68)

GCGCCTCGTAGGTCACAGTT

(SLACS forward and reverse primers, respectively),

wherein steps (a) through (f) are repeated using at least one nucleic acid primer pair that is different for each Leishmania species in the group.

2. The method of claim 1 , further comprising after performing temperature cycles, determining the quantity of Leishmania in the sample comprising the steps of (i) determining the time or number of temperature cycles at which a threshold level of double-stranded DNA product and/or pyrophosphate product is reached; and (ii) comparing the time or number of temperature cycles of (i) to a reference standard, so as to quantify the amount of Leishmania in the sample.

3. The method of claim 2 , wherein the threshold level of double-stranded DNA product is determined for a single copy gene, and wherein the reference standard is a reference standard curve generated with different known amount of a particular Leishmania species.

4. The method of claim 2 , wherein the reference standard is a reference standard curve.

5. The method of claim 1 , wherein determining the melting temperature comprises using ultraviolet absorption detection, fluorescence detection, light scattering detection, colorimetric detection, or chromogenic detection.

6. The method of claim 1 , wherein steps (a) to (d) are conducted in a single reaction vessel, well, or tube.