Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Leishmania Strains, Clinical Samples and DNA Extraction
2.2. ITS1-PCR RFLP
2.3. DNA Sequencing and Phylogenetic Analysis
2.4. qPCR Assays
2.5. High-Resolution Melt (HRM) Analysis
2.6. Ethics Approval
2.7. Statistical Analysis
3. Results
3.1. Both L. (L.) mexicana and L. (L.) amazonensis Can be Distinguished from L. (L.) infantum Exploiting A Differential qPCR Targeting Minicircle kDNA
3.2. L. (L.) amazonensis Can be Differentiated from L. (L.) mexicana by qPCR-ITS1 HRM Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Species | Strain/Isolate |
---|---|
L. (L.) infantum | MHOM/TN/80/IPT1 |
L. (L.) infantum | MHOM/IT/86/ISS218 |
L. (L.) infantum | MHOM/FR/78/LEM75 |
L. (L.) amazonensis | MHOM/BR/00/LTB0016 |
L. (L.) amazonensis | IFLA/BR/67/PH8 |
L. (L.) amazonensis | Clinical isolate |
L. (V.) braziliensis | MHOM/BR/75/M2904 |
L. (L.) mexicana | MHOM/MX/2011/Lacandona |
L. (L.) mexicana | Clinical isolate 2 |
L. (L.) mexicana | Clinical isolate 3 |
L. (L.) mexicana | Clinical isolate 5 |
L. (L.) mexicana | Clinical isolate 14 |
L. (L.) mexicana | Clinical isolate 17 |
Sample | Species Identification (ITS1-PCR RFLP) | Species Identification (ALAT sequencing) | CL Form |
---|---|---|---|
Px1 | L. (L.) mexicana | n.a. 1 | LCL |
Px2 | L. (L.) mexicana | L. (L.) mexicana | LCL |
Px3 | L. (L.) mexicana | L. (L.) mexicana | LCL |
Px4 | L. (L.) mexicana | n.a. 1 | LCL |
Px5 | L. (L.) mexicana | n.a. 1 | DCL |
Px7 | L. (L.) mexicana | n.a. 1 | LCL |
Px9 | L. (L.) mexicana | L. (L.) mexicana | DCL |
Px10 | L. (L.) mexicana | L. (L.) mexicana | LCL |
PxGSF | L. (L.) mexicana | n.a. 1 | LCL |
PxCMU | n.a. 1 | n.a. 1 | n.a. 1 |
PxJLC | L. (L.) mexicana | L. (L.) mexicana | LCL |
Leishmania Species, Strain/Isolate | DNA Template (ng) | qPCR-ML (Cq ± SD) | qPCR-ama (Cq ± SD) |
---|---|---|---|
L. (L.)mexicana MHOM/MX/2011/Lacandona | 1.0 | 31.61 ± 2.03 | 14.25 ± 0.69 |
L. (L.)mexicana MHOM/MX/2011/Lacandona | 1.0 × 10−1 | 33.43 ± 2.09 | 17.48 ± 1.13 |
L. (L.)mexicana MHOM/MX/2011/Lacandona | 1.0 × 10−2 | 37.53 ± 1.27 | 20.68 ± 1.46 |
L. (L.)mexicana MHOM/MX/2011/Lacandona | 1.0 × 10−3 | n.d. 1 | 24.07 ± 1.09 |
L. (L.)mexicana MHOM/MX/2011/Lacandona | 1.0 × 10−4 | n.d. 1 | 27.37 ± 1.05 |
L. (L.)mexicana MHOM/MX/2011/Lacandona | 1.0 × 10−5 | n.d.1 | 31.36 ± 0.93 |
L. (L.)mexicana Isolate 2 | 1.0 | 33.19 ± 1.34 | 16.78 ± 0.06 |
L. (L.)mexicana Isolate 3 | 1.0 | 33.62 ± 2.14 | 18.62 ± 1.12 |
L. (L.)mexicana Isolate 5 | 1.0 | 38.19 ± 1.01 | 20.14 ± 0.43 |
L. (L.)mexicana Isolate 14 | 1.0 | 34.59 ± 0.51 | 16.54 ± 0.17 |
L. (L.)mexicana Isolate 17 | 1.0 | 35.94 ± 1.20 | 19.15 ± 1.00 |
L. (L.)amazonensis MHOM/BR/00/LTB0016 | 1.0 × 10−1 | 33.95 ± 0.34 | 21.1 ±1.02 |
L. (L.)infantum MHOM/FR/78/LEM75 | 1.0 | 14.42 ± 0.75 | 28.02 ± 0.98 |
Sample ID | qPCR-ML (Cq ± SD) | qPCR-ama (Cq ± SD) |
---|---|---|
Px1 | n.d. 1 | 27.71 ± 0.08 |
Px2 | n.d. 1 | 25.79 ± 0.55 |
Px3 | n.d. 1 | 24.57 ± 0.58 |
Px4 | 36.21 ± 1.99 | 29.59 ± 0.80 |
Px5 | n.d. 1 | 28.99 ± 1.57 |
Px7 | 35.31 ± 1.47 | 28.04 ± 0.25 |
Px9 | n.d. 1 | 28.35 ± 1.93 |
Px10 | n.d. 1 | 24.92 ± 0.87 |
PxGSF | 36.38 ± 1.70 | 29.49 ± 0.53 |
PxCMU | 36.53 ± 1.36 | 31.48 ± 0.36 |
PxJLC | n.d. 1 | 34.87 ± 1.93 |
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Ceccarelli, M.; Diotallevi, A.; Buffi, G.; De Santi, M.; Fernández-Figueroa, E.A.; Rangel-Escareño, C.; Muñoz-Montero, S.A.; Becker, I.; Magnani, M.; Galluzzi, L. Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis. Microorganisms 2020, 8, 818. https://doi.org/10.3390/microorganisms8060818
Ceccarelli M, Diotallevi A, Buffi G, De Santi M, Fernández-Figueroa EA, Rangel-Escareño C, Muñoz-Montero SA, Becker I, Magnani M, Galluzzi L. Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis. Microorganisms. 2020; 8(6):818. https://doi.org/10.3390/microorganisms8060818
Chicago/Turabian StyleCeccarelli, Marcello, Aurora Diotallevi, Gloria Buffi, Mauro De Santi, Edith A. Fernández-Figueroa, Claudia Rangel-Escareño, Said A. Muñoz-Montero, Ingeborg Becker, Mauro Magnani, and Luca Galluzzi. 2020. "Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis" Microorganisms 8, no. 6: 818. https://doi.org/10.3390/microorganisms8060818
APA StyleCeccarelli, M., Diotallevi, A., Buffi, G., De Santi, M., Fernández-Figueroa, E. A., Rangel-Escareño, C., Muñoz-Montero, S. A., Becker, I., Magnani, M., & Galluzzi, L. (2020). Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis. Microorganisms, 8(6), 818. https://doi.org/10.3390/microorganisms8060818