Supplementary File S1. Output from the NCBI Primer-BLAST tool showing the in silico BLAST results for the LEISH primers and the TaqMan® MGB probe and also GIO set (primers and probe). The file includes alignment details, specificity analysis, and confirmation of exclusive matches with Leishmania infantum/chagasi sequences, with no cross-reactivity detected for other Trypanosomatidae species.

RID: 29RP14B8013	
Job Title: LEISH GIO				
Program: BLASTN 
Database: core_nt Core nucleotide BLAST database
Query #1: LEISH-1 Query ID: lcl|Query_6951190 Length: 23

Sequences producing significant alignments:
Description                                                       Name            Name            Taxid      Score  Score cover Value Ident  Len        Accession        
Leishmania infantum isolate ZK25-class7 minicircle kinetoplast... Leishmania i... NA              5671       46.1   46.1  100%  0.023 100.00 689        MT598576.1       
Leishmania donovani strain MHOM/SD/97/LEM3475 minicircle DNA...   Leishmania d... NA              5661       46.1   46.1  100%  0.023 100.00 758        KY950651.1       
Leishmania infantum isolate ZK47-class10 minicircle kinetoplas... Leishmania i... NA              5671       46.1   46.1  100%  0.023 100.00 726        MT598268.1       
Leishmania infantum isolate ZK47-class3 minicircle kinetoplast... Leishmania i... NA              5671       46.1   46.1  100%  0.023 100.00 705        MT598379.1       
Leishmania infantum isolate ZK47-class41 minicircle kinetoplas... Leishmania i... NA              5671       46.1   46.1  100%  0.023 100.00 703        MT598457.1       


Query #2: LEISH-2 Query ID: lcl|Query_6951191 Length: 17

Sequences producing significant alignments:
Description                                                       Name            Name            Taxid      Score  Score cover Value Ident  Len        Accession        
Leishmania donovani voucher VCRC-CL77 minicircle kinetoplast...   Leishmania d... NA              5661       34.2   34.2  100%  88    100.00 554        PP664458.1       
PREDICTED: Haliotis rufescens solute carrier family 13 member...  Haliotis ruf... red abalone     6454       34.2   34.2  100%  88    100.00 2957       XM_048396497.1   
Leishmania donovani voucher VCRC-CL79 minicircle kinetoplast...   Leishmania d... NA              5661       34.2   34.2  100%  88    100.00 550        PP664453.1       
PREDICTED: Mus musculus zinc finger and BTB domain containing ... Mus musculus    house mouse     10090      34.2   34.2  100%  88    100.00 5654       XM_006539804.4   
Leishmania infantum isolate MWD1 minicircle kinetoplast sequence  Leishmania i... NA              5671       34.2   34.2  100%  88    100.00 140        MK697545.1       

Query #3: Probe Taqman MGB Query ID: lcl|Query_6951192 Length: 18

Sequences producing significant alignments:
Description                                                       Name            Name            Taxid      Score  Score cover Value Ident  Len        Accession        
Vibrio alginolyticus strain CAB15 chromosome 1                    Vibrio algin... NA              663        36.2   36.2  100%  22    100.00 3347710    CP167876.1       
Vibrio cholerae strain SPH 24-01 chromosome 1, complete sequence  Vibrio cholerae NA              666        36.2   36.2  100%  22    100.00 2894403    CP173026.1       
Bdellovibrio bacteriovorus strain Bd16 chromosome, complete...    Bdellovibrio... NA              959        36.2   36.2  100%  22    100.00 3431168    CP168971.1       
PREDICTED: Euwallacea fornicatus neuroligin-1-like...             Euwallacea f... NA              995702     36.2   36.2  100%  22    100.00 5144       XM_066287820.1   
Vibrio sp. SCSIO 43097 chromosome 1, complete sequence            Vibrio sp. S... NA              2819104    36.2   36.2  100%  22    100.00 3417782    CP071840.1       


RID: 29RP14B8013	
Job Title: GIO 				
Program: BLASTN 
Database: core_nt Core nucleotide BLAST database


Query #4: GIO-06F Query ID: lcl|Query_6951193 Length: 20

Sequences producing significant alignments:
Description                                                       Name            Name            Taxid      Score  Score cover Value Ident  Len        Accession        
Leishmania donovani isolate MHOM/KE/73/MRC74 kinetoplast...       Leishmania d... NA              5661       40.1   40.1  100%  1.4   100.00 806        AF239703.1       
L.infantum (AJS-IPTBG) kinetoplast DNA                            Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 770        Z35269.1         
Leishmania infantum isolate MCAN/CN/90/SC clone III minicircle... Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 116        HQ585885.1       
Leishmania donovani isolate MHOM/KE/73/MRC74 kinetoplast...       Leishmania d... NA              5661       40.1   40.1  100%  1.4   100.00 794        AF167717.1       
Leishmania infantum isolate ZK5-class7 minicircle kinetoplast...  Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 741        MT598578.1       

Query #5: GIO-96P Query ID: lcl|Query_6951194 Length: 20

Sequences producing significant alignments:                                                                  Scientific      Common                     Max    Total Query   E   Per.   Acc.                        
Description                                                       Name            Name            Taxid      Score  Score cover Value Ident  Len        Accession        
Leishmania infantum isolate ZK5-class7 minicircle kinetoplast...  Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 741        MT598578.1       
Leishmania infantum isolate ZK47-class7 minicircle kinetoplast... Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 733        MT598579.1       
Leishmania infantum isolate LIPA83-class7 minicircle kinetopla... Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 730        MT598575.1       
PREDICTED: Oncorhynchus kisutch uncharacterized LOC116375907...   Oncorhynchus... coho salmon     8019       38.2   38.2  95%   5.6   100.00 3124       XM_031834532.1   
PREDICTED: Oncorhynchus kisutch uncharacterized LOC116375907...   Oncorhynchus... coho salmon     8019       38.2   38.2  95%   5.6   100.00 3657       XM_031834531.1   

Query #6: GIO-155R Query ID: lcl|Query_6951195 Length: 20

Sequences producing significant alignments:
Description                                                       Name            Name            Taxid      Score  Score cover Value Ident  Len        Accession        
PREDICTED: Equus przewalskii ubiquitin conjugating enzyme E2 U... Equus przewa... Przewalski's... 9798       40.1   40.1  100%  1.4   100.00 2068       XR_011532552.1   
Leishmania infantum isolate ZK5-class7 minicircle kinetoplast...  Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 741        MT598578.1       
Leishmania infantum isolate ZK47-class7 minicircle kinetoplast... Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 733        MT598579.1       
PREDICTED: Equus quagga ubiquitin conjugating enzyme E2 U...      Equus quagga    NA              89248      40.1   40.1  100%  1.4   100.00 2598       XR_006885981.1   
Leishmania infantum isolate LIPA83-class7 minicircle kinetopla... Leishmania i... NA              5671       40.1   40.1  100%  1.4   100.00 730        MT598575.1       
