title: Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years.
abstract: Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.
authors.affiliation: Pathology Department, Medical School of São Paulo University, São Paulo, Brazil. carolstocco@gmail.com
authors.forename: Ana Carolina S
authors.initials: AC
authors.lastname: de Lima
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Zampieri
authors.forename: Thaíse Y
authors.initials: TY
authors.lastname: Tomokane
authors.forename: Márcia D
authors.initials: MD
authors.lastname: Laurenti
authors.forename: Fernando T
authors.initials: FT
authors.lastname: Silveira
authors.forename: Carlos E P
authors.initials: CE
authors.lastname: Corbett
authors.forename: Lucile M
authors.initials: LM
authors.lastname: Floeter-Winter
authors.forename: Cláudia M C
authors.initials: CM
authors.lastname: Gomes
mesh_terms: D001483:Base Sequence; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D007891:Leishmania / Q000235:genetics* / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012335:RNA, Ribosomal / Q000235:genetics; D054679:Ribosome Subunits, Small; D016415:Sequence Alignment; D012867:Skin / Q000469:parasitology; D013997:Time Factors
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal