Table 3.
Relative efficiencies of SYBR green or TaqMan assays for detecting and differentiating Leishmania spp. or Leptomonas spp. or Crithidia fasciculata
| Type of PCR assay, primer set designation | Avg CT valuea |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| L. (L.) amazonensis | L. (V.) braziliensis | L. (L.) chagasi | L. (L.) donovani | L. (L.) infantum | L. (L.) major | L. (L.) mexicana | L. (L.) tropica | Leptomonas | Crithidia | |
| SYBR green | ||||||||||
| Alpha-tubulin | 21.4 | >30 | 20.7 | 20.3 | 21.1 | 20.8 | 21.4 | 23.4 | 27.8 | 28.3 |
| Cytochrome B 1 | >30 | >30 | 23.8 | 20.0 | 22.0 | >30 | >30 | >30 | >30 | >30 |
| DNA polymerase 1 | 25.1 | 29.0 | 23.5 | 23.0 | 24.7 | 24.5 | 25.6 | 27.5 | >30 | >30 |
| DNA polymerase 2 | 24.1 | 23.4 | 22.9 | 22.5 | 24.3 | 24.2 | 24.7 | 27.9 | 29.2 | 26.5 |
| GPI | 24.3 | 29.4 | 23.1 | 22.9 | 24.4 | 24.6 | 25.2 | 27.1 | >30 | >30 |
| HSP70-1 | 21.3 | 21.4 | 19.8 | 19.4 | 21.3 | 20.8 | 22.5 | 23.7 | >30 | >30 |
| HSP70-4 | 21.7 | 22.1 | 20.1 | 19.9 | 21.5 | 20.9 | 22.8 | 24.0 | 27.0 | 23.6 |
| kDNA 1 | 15.6 | 27.2 | 15.7 | 14.4 | 15.3 | 19.6 | 16.5 | 15.4 | 26.6 | 13.2 |
| kDNA 2 | >30 | 29.3 | 14.6 | 12.8 | 14.4 | 11.8 | 19.2 | 29.8 | 26.4 | 29.6 |
| kDNA 3 | 14.7 | 18.9 | 14.8 | 13.0 | 14.3 | 11.4 | 13.7 | 15.4 | 23.4 | 18.3 |
| kDNA 4 | 16.3 | 25.6 | 14.6 | 12.9 | 14.4 | 11.7 | 14.8 | 21.5 | 26.0 | 27.8 |
| kDNA 5 | >30 | 28.7 | 14.8 | 13.1 | 14.3 | 11.6 | 20.3 | 19.0 | 26.7 | 26.5 |
| kDNA 7 | 19.8 | 28.9 | 14.8 | 12.6 | 14.2 | 11.5 | 18.6 | 23.9 | 25.6 | 29.4 |
| L. (L.) amazonensis cytochrome B 1 | 28.4 | 25.6 | 29.1 | 29.8 | 27.4 | >30 | >30 | >30 | >30 | >30 |
| L. (L.) amazonensis DNA polymerase 1 | 24.4 | 23.2 | 23.4 | 22.9 | 24.8 | 25.4 | 24.7 | 28.0 | 29.1 | 25.5 |
| L. (L.) amazonensis kDNA 3 | 15.6 | 27.0 | 15.6 | 14.5 | 15.2 | 19.3 | 16.5 | 15.2 | 27.2 | 13.1 |
| L. (L.) amazonensis kDNA 4 | 15.9 | >30 | >30 | >30 | >30 | >30 | 19.0 | >30 | >30 | >30 |
| L. (L.) amazonensis kDNA 2 | 15.2 | >30 | >30 | 28.2 | 29.5 | 24.3 | 21.8 | >30 | >30 | >30 |
| L. (L.) amazonensis kDNA 1 | 15.0 | 29.8 | >30 | 27.9 | >30 | 23.8 | 21.6 | >30 | >30 | >30 |
| L. (V.) braziliensis DNA polymerase 1 | >30 | 22.9 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | >30 |
| L. (V.) braziliensis DNA polymerase 2 | 28.1 | 23.1 | 24.3 | 23.7 | 25.5 | >30 | 28.6 | 28.3 | 26.6 | >30 |
| L. (V.) braziliensis kDNA 1 | >30 | 13.5 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | >30 |
| L. (V.) braziliensis kDNA 2 | >30 | 23.9 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | >30 |
| L. (V.) braziliensis kDNA 3 | >30 | 17.7 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | >30 |
| L. (L.) chagasi SIDER 1 | >30 | >30 | 21.7 | 21.7 | 22.4 | >30 | >30 | 26.4 | >30 | >30 |
| L. (L.) infantum minicircle 1 | >30 | >30 | 23.8 | >30 | 26.6 | >30 | >30 | >30 | >30 | >30 |
| L. major MAG 1 | 23.2 | 27.6 | 22.4 | 21.3 | 24.5 | 24.1 | 25.1 | 23.2 | >30 | >30 |
| L. (L.) major minicircle 1 | 18.6 | 29.4 | 16.1 | 22.6 | 15.9 | 22.6 | 18.9 | >30 | 25.9 | 29.9 |
| L. (L.) mexicana minicircle 1 | >30 | >30 | >30 | >30 | >30 | >30 | 20.1 | >30 | >30 | >30 |
| L. (L.) tropica cytochrome B 1 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | 25.1 | >30 | >30 |
| L. (L.) tropica cytochrome B 2 | >30 | >30 | 24.6 | 26.2 | 22.7 | >30 | >30 | 24.3 | >30 | >30 |
| L. (L.). tropica cytochrome B 3 | 28.4 | >30 | >30 | 29.4 | >30 | 23.2 | >30 | 26.2 | >30 | >30 |
| L. (L.) tropica cytochrome B 4 | 24.1 | >30 | 26.7 | 23.2 | 24.8 | 22.0 | 24.5 | 25.0 | >30 | >30 |
| L. (L.) tropica minicircle 1 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | 19.8 | >30 | >30 |
| L. (L.) donovani minicircle 1 | >30 | >30 | 29.4 | 15.3 | 25.9 | >30 | >30 | >30 | >30 | >30 |
| Leptomonas GAPDH 2 | >30 | 24.1 | >30 | >30 | >30 | >30 | >30 | >30 | 26.6 | 23.5 |
| Leptomonas miniexon 1 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | 20.7 | >30 |
| MAG 1 | >30 | >30 | 21.2 | 20.8 | 23.6 | >30 | >30 | 25.8 | >30 | >30 |
| MAG 2 | >30 | >30 | 21.0 | 20.8 | 23.4 | >30 | >30 | >30 | >30 | >30 |
| Maxicircle 1 | >30 | >30 | 22.3 | 19.6 | 19.2 | >30 | >30 | >30 | >30 | >30 |
| Miniexon 1 | 21.6 | 26.3 | 17.9 | 16.7 | 19.6 | 19.0 | 25.5 | 18.9 | 29.6 | 29.1 |
| Miniexon 2 | 29.9 | 16.9 | 29.0 | 29.9 | 29.3 | 29.3 | 30.0 | >30 | >30 | >30 |
| SLACS | 29.9 | 19.7 | 29.8 | 30.0 | 29.9 | >30 | 29.9 | >30 | >30 | 29.8 |
| TaqMan | ||||||||||
| TaqMan alpha-tubulin | 26.1 | >30 | 25.0 | 24.9 | 25.9 | 24.7 | 26.2 | 28.8 | >30 | >30 |
| TaqMan cytochrome B 1 | >30 | >30 | 27.7 | 24.5 | 26.1 | >30 | >30 | >30 | >30 | >30 |
| TaqMan DNA polymerase 1 | 27.1 | >30 | 25.8 | 25.1 | 27.3 | 26.7 | 27.8 | 29.4 | >30 | >30 |
| TaqMan DNA polymerase 2 | 27.0 | 26.7 | 26.5 | 26.0 | 27.9 | 26.9 | 27.9 | 28.8 | >30 | >30 |
| TaqMan kDNA 4 | >30 | >30 | 18.1 | 17.3 | 17.8 | >30 | >30 | >30 | >30 | >30 |
| TaqMan kDNA 5 | >30 | >30 | 18.1 | 16.1 | 18.0 | 16.1 | 25.5 | 25.3 | >30 | >30 |
| TaqMan kDNA 7 | >30 | >30 | 18.4 | 17.5 | 18.1 | 23.6 | >30 | >30 | >30 | >30 |
| TaqMan L. (V.) braziliensis kDNA 3 | >30 | 21.9 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | >30 |
| TaqMan L. (L.) infantum minicircle 1 | >30 | >30 | 28.8 | >30 | >30 | >30 | >30 | >30 | >30 | >30 |
| TaqMan L. (L.) tropica minicircle 1 | >30 | >30 | >30 | >30 | >30 | >30 | >30 | 23.6 | >30 | >30 |
| TaqMan L. (L.) donovani minicircle 1 | >30 | >30 | >30 | 18.1 | >30 | >30 | >30 | >30 | >30 | >30 |
| TaqMan MAG 1 | >30 | >30 | 23.5 | 24.3 | 25.9 | >30 | >30 | 28.3 | >30 | >30 |
| TaqMan MAG 2 | >30 | >30 | 23.8 | 23.5 | 26.1 | >30 | >30 | >30 | >30 | >30 |
| TaqMan maxicircle 1 | >30 | >30 | 26.7 | 25.0 | 24.2 | >30 | >30 | >30 | >30 | >30 |
| TaqMan miniexon 1 | 23.8 | >30 | 26.5 | 24.0 | 26.8 | 22.0 | >30 | 22.9 | >30 | >30 |
| Multiplex TaqMan | ||||||||||
| TaqMan MAG 2; 5′FAM/3′TAMRA | >30 | >30 | 23.7 | 23.4 | 26.0 | >30 | >30 | >30 | >30 | >30 |
| TaqMan DNA polymerase 2; 5′TET /3′Iowa Black | 25.3 | 24.9 | 25.7 | 24.9 | 26.6 | 25.6 | 25.9 | 28.8 | >30 | >30 |
a
The data indicate the average CT values of 2 replicates for Leptomonas and L. amazonensis or of 4 or more replicates for all other species. CT values reflect the cycle in which fluorescence intensity reached 0.2 when amplifying 0.1 ng of total parasite DNA in the presence of 1 ng of human DNA. The cutoff of CT = 30 avoids ambiguities caused by primer-dimers in SYBR green assays. Please note that CT cutoffs could differ between different strains or when assays are performed in different laboratories.