Fig 1.
Development and interpretation of the duplex PCR-LFD for detecting Leishmania martiniquensis and Leishmania orientalis.
(A) Schematic representation of the duplex PCR-LFD. The test strip consisted of specific test lines for capturing amplicons of L. martiniquensis (Test line 1) and L. orientalis (Test line 2), with a control line to ensure assay validity. (B) The test strips are representative of positive and negative results. The specific bands corresponding to the presence of L. orientalis (L. o positive), L. martiniquensis (L. m positive), and both L. orientalis and L. martiniquensis (L. o/L. m positive) amplicons are shown. For the absence of any target amplicons, only the control line is observed (negative).
Fig 2.
Specificity of the duplex PCR and PCR-LFD for identification of L. martiniquensis and L. orientalis.
(A) Singleplex PCR and duplex PCR reactions. M: 100 bp DNA ladder; Lane 1–2: Singleplex PCR using universal ITS-F and Lm-ITS-288/308R tested with L. martiniquensis and L. orientalis DNA templates, respectively. Lane 3–4: Singleplex PCR using universal ITS-F and Ls-ITS-233/250R tested with DNA templates from L. orientalis and L. martiniquensis, respectively. Lane 5–7: Duplex PCR reactions tested with DNA templates from L. martiniquensis, L. orientalis and mixed DNA templates from both species. N: Non-template control. (B) and (C) Cross-reactivity of duplex PCR and PCR-LFD, respectively; Lane 1–16: L. martiniquensis (1), L. orientalis (2), L. donovani (3), L. infantum (4), Streptococcus pyogenes (5), Entamoeba sp. (6), Neisseria gonorrhoeae (7), Escherichia coli (8), Salmonella typhi (9), Corynebacterium diphtheria (10), Plasmodium falciparum (11), Vibrio cholera (12), Trichomonas hominis (13), Shigella flexneri (14), Giardia duodenalis (15), Human (16). N: Non-template control.
Fig 3.
Limits of duplex PCR and PCR-LFD detection based on genomic DNA of L. martiniquensis and L. orientalis.
(A) Limit of detection of duplex PCR using L. martiniquensis DNA. (B) Limit of detection of duplex PCR using L. orientalis DNA. (C) Limit of detection of duplex PCR using a mixture of L. martiniquensis and L. orientalis DNA. Lanes 1–9 of (A-C): the amount of DNA template was 1000, 500, 100, 50, 10, 5, 1, 0.5, and 0.1 parasite/μL, respectively. N: Non-template control. (D) Limit of detection of PCR-LFD using tenfold serially diluted DNA templates (105−10−5 parasite/μL) including L. martiniquensis DNA (top panel), L. orientalis DNA (middle panel), and 1:1 ratio mixture of L. martiniquensis and L. orientalis DNA (bottom panel).
Table 1.
Sensitivity and specificity of PCR-LFD assay for duplex detection of L. martiniquensis and L. orientalis in asymptomatic patients with HIV.