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NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION.

Gualda KP, Marcussi LM, Neitzke-Abreu HC, Aristides SM, Lonardoni MV, Cardoso RF, Silveira TG - Rev. Inst. Med. Trop. Sao Paulo (2015 Sep-Oct)

Bottom Line: Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed.In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative.The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

View Article: PubMed Central - PubMed

Affiliation: Universidade Estadual de Maringá, Maringá, PR, Brasil.

ABSTRACT
Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

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- Gel showing the specificity of the 230 bp fragment of the kDNA of Leishmania infantum in clinical samples. The PCR with FLC2/RLC2 primers was carried out using as template the DNA extracted from clinical samples. kDNA, kinetoplast DNA; M, 100 bp molecular marker. Panel A. Lanes 1, 2, 3, 4 and 5: DNA from lesions in different humans with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6: L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel B. Lane 1: DNA from the lesion of a dog with clinical suspicion of VL; Lane 2: DNA from the blood sample of a dog clinically suspected to have VL; Lanes 3, 4 and 5: DNA from lesions of different dogs with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6:L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel C.Lane 1: DNA from promastigotes isolated from a hamster inoculated with tissues from the lesion of a dog with clinical suspicion of VL (Isolate I); Lane 2: DNA from promastigotes isolated from a hamster inoculated with the blood sample of a dog with clinical suspicion of VL (Isolate II); Lane 3: L. infantum (MCAN/ BR/2010/CUR392); Lane 4: L. infantum(MHOM/BR/1974/PP75).
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f04: - Gel showing the specificity of the 230 bp fragment of the kDNA of Leishmania infantum in clinical samples. The PCR with FLC2/RLC2 primers was carried out using as template the DNA extracted from clinical samples. kDNA, kinetoplast DNA; M, 100 bp molecular marker. Panel A. Lanes 1, 2, 3, 4 and 5: DNA from lesions in different humans with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6: L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel B. Lane 1: DNA from the lesion of a dog with clinical suspicion of VL; Lane 2: DNA from the blood sample of a dog clinically suspected to have VL; Lanes 3, 4 and 5: DNA from lesions of different dogs with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6:L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel C.Lane 1: DNA from promastigotes isolated from a hamster inoculated with tissues from the lesion of a dog with clinical suspicion of VL (Isolate I); Lane 2: DNA from promastigotes isolated from a hamster inoculated with the blood sample of a dog with clinical suspicion of VL (Isolate II); Lane 3: L. infantum (MCAN/ BR/2010/CUR392); Lane 4: L. infantum(MHOM/BR/1974/PP75).

Mentions: contamination or reaction inhibition (Fig. 3). Analysis of DNA samples from different human patients with a confirmed diagnosis (Fig. 4A) of cutaneous leishmaniasis indicated that the primers showed specificity for L. infantum.Figure 4B shows the specificity of the primers in DNA samples from lesions of dogs diagnosed with cutaneous leishmaniasis (parasitological, serological and molecular diagnoses) and a DNA sample from the blood and lesion of a dog with clinically suspected VL


NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION.

Gualda KP, Marcussi LM, Neitzke-Abreu HC, Aristides SM, Lonardoni MV, Cardoso RF, Silveira TG - Rev. Inst. Med. Trop. Sao Paulo (2015 Sep-Oct)

- Gel showing the specificity of the 230 bp fragment of the kDNA of Leishmania infantum in clinical samples. The PCR with FLC2/RLC2 primers was carried out using as template the DNA extracted from clinical samples. kDNA, kinetoplast DNA; M, 100 bp molecular marker. Panel A. Lanes 1, 2, 3, 4 and 5: DNA from lesions in different humans with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6: L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel B. Lane 1: DNA from the lesion of a dog with clinical suspicion of VL; Lane 2: DNA from the blood sample of a dog clinically suspected to have VL; Lanes 3, 4 and 5: DNA from lesions of different dogs with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6:L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel C.Lane 1: DNA from promastigotes isolated from a hamster inoculated with tissues from the lesion of a dog with clinical suspicion of VL (Isolate I); Lane 2: DNA from promastigotes isolated from a hamster inoculated with the blood sample of a dog with clinical suspicion of VL (Isolate II); Lane 3: L. infantum (MCAN/ BR/2010/CUR392); Lane 4: L. infantum(MHOM/BR/1974/PP75).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660445&req=5

f04: - Gel showing the specificity of the 230 bp fragment of the kDNA of Leishmania infantum in clinical samples. The PCR with FLC2/RLC2 primers was carried out using as template the DNA extracted from clinical samples. kDNA, kinetoplast DNA; M, 100 bp molecular marker. Panel A. Lanes 1, 2, 3, 4 and 5: DNA from lesions in different humans with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6: L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel B. Lane 1: DNA from the lesion of a dog with clinical suspicion of VL; Lane 2: DNA from the blood sample of a dog clinically suspected to have VL; Lanes 3, 4 and 5: DNA from lesions of different dogs with confirmed diagnosis of cutaneous leishmaniasis (parasitological, serological and molecular diagnoses); Lane 6:L. braziliensis (MHOM/BR/1987/M11272); Lane 7:L. infantum (MHOM/BR/1974/PP75). Panel C.Lane 1: DNA from promastigotes isolated from a hamster inoculated with tissues from the lesion of a dog with clinical suspicion of VL (Isolate I); Lane 2: DNA from promastigotes isolated from a hamster inoculated with the blood sample of a dog with clinical suspicion of VL (Isolate II); Lane 3: L. infantum (MCAN/ BR/2010/CUR392); Lane 4: L. infantum(MHOM/BR/1974/PP75).
Mentions: contamination or reaction inhibition (Fig. 3). Analysis of DNA samples from different human patients with a confirmed diagnosis (Fig. 4A) of cutaneous leishmaniasis indicated that the primers showed specificity for L. infantum.Figure 4B shows the specificity of the primers in DNA samples from lesions of dogs diagnosed with cutaneous leishmaniasis (parasitological, serological and molecular diagnoses) and a DNA sample from the blood and lesion of a dog with clinically suspected VL

Bottom Line: Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed.In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative.The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

View Article: PubMed Central - PubMed

Affiliation: Universidade Estadual de Maringá, Maringá, PR, Brasil.

ABSTRACT
Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

Show MeSH
Related in: MedlinePlus