Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia
Abstract
:1. Introduction
2. Material and Methods
2.1. Search Strategy for New Gene Targets and Primer Design
2.2. DNA Extraction
2.3. Real-Time Quantitative PCR (qPCR) Assays
2.3.1. Standard Curve
2.3.2. qPCR Assay Evaluation
2.4. PCR, Amplicon Sequencing, and Sequence Analysis
2.5. Experimental Validation
2.5.1. In Vitro Infection with THP-1 Macrophages
2.5.2. In Vivo Infection
2.5.3. Sample Collection
2.6. Data Analysis
2.7. Ethics Statement
3. Results
3.1. Real-Time PCR Performance and Validation of the Method
3.2. Validation of the Novel qPCR Assay and Gene Targets in Experimental Conditions
3.3. Validation of the Novel qPCR Assay and Gene Targets through Parasite Detection in Clinical Isolate Cultures and Host Tissue Samples
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Primer F (5′-3′) | Primer R (5′-3′) | Amplicon Size | Usage |
---|---|---|---|---|
LinJ31seq | GTGCACGCCAATGTCTTTGT | GCCCATGGTTGAGCTAGGTT | 444 bp | Detection of L. infantum by PCR |
LinJ31_2420 | GCAGCGATGCGACCTAGTTA | TGTACAAGGCACTGGCGTAG | 98 bp | Detection and quantification of L. infantum by qPCR |
Crid2.1seq | TCACTTTGGCGGTATCAGTG | GCATCAGCTGACCCTTTCTC | 502 bp | Detection of Crithidia sp. LVH60A and C. fasciculata by PCR |
LVH60a_Tig001 | GTTAGAGCGACTAGCCCGTG | GGGTAGAGGAGAGAGGTGGG | 128 bp | Detection of Crithidia sp. LVH60A by PCR |
Catalase_LVH60-12060_1F | TCAGCGAGTCGGAGTCTAA | CGAATAAGGGTGGAAACAAAGAG | 107 bp | Detection and quantification of Crithidia sp. LVH60A and C. fasciculata by qPCR |
Primer | Species | Chr. | Gene ID or Access Number * | Amp. ** GC% | Amp. Size | Percent Nucleotide Identity |
---|---|---|---|---|---|---|
Crid2.1seq | Crithidia fasciculata (CfCl) | 16 | CFAC1_160022800 | 67.47% | 501 bp | A = 87.0 T = 76.0 C = 162.0 G = 176.0 |
Crithidia sp. LVH60A (LVH60a) | 10 | CP119667.1 | 67.13% | 502 bp | A = 86.0 T = 79.0 C = 166.0 G = 171.0 | |
Leptomonas pyrrhocoris (H10) | 15 | LpyrH10_15 a | No match | |||
16 | LpyrH10_16 b | |||||
Leptomonas seymouri (ATCC_30220) | 48 | Lsey_0048 a | No match | |||
01 | Lsey_01 b |
Tissue | Number of Samples | PCR + LinJ31seq | PCR + Crid2.1seq | Nested-PCR TRY927/SSU561 [57] | |
---|---|---|---|---|---|
Patient samples | BMVL a | 18 | 13 | 0 c | 18 |
PB | 14 | 6 | 0 | 7 | |
Clinical isolates | CI b | 53 | 14 | 48 | 46 |
Skin (nodule/papule) | 4 | 2 | 2 | 4 c | |
Spleen | 1 | 0 | 1 | 1 d |
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Takamiya, N.T.; Rogerio, L.A.; Torres, C.; Leonel, J.A.F.; Vioti, G.; de Sousa Oliveira, T.M.F.; Valeriano, K.C.; Porcino, G.N.; de Miranda Santos, I.K.F.; Costa, C.H.N.; et al. Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia. Trop. Med. Infect. Dis. 2023, 8, 405. https://doi.org/10.3390/tropicalmed8080405
Takamiya NT, Rogerio LA, Torres C, Leonel JAF, Vioti G, de Sousa Oliveira TMF, Valeriano KC, Porcino GN, de Miranda Santos IKF, Costa CHN, et al. Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia. Tropical Medicine and Infectious Disease. 2023; 8(8):405. https://doi.org/10.3390/tropicalmed8080405
Chicago/Turabian StyleTakamiya, Nayore Tamie, Luana Aparecida Rogerio, Caroline Torres, João Augusto Franco Leonel, Geovanna Vioti, Tricia Maria Ferreira de Sousa Oliveira, Karoline Camila Valeriano, Gabriane Nascimento Porcino, Isabel Kinney Ferreira de Miranda Santos, Carlos H. N. Costa, and et al. 2023. "Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia" Tropical Medicine and Infectious Disease 8, no. 8: 405. https://doi.org/10.3390/tropicalmed8080405