Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3' untranslated region of the heat shock protein 70 (type I) gene.
PLoS Negl Trop Dis
; 15(11): e0009982, 2021 11.
Article
in En
| MEDLINE
| ID: mdl-34847144
ABSTRACT
PCR-based methods to amplify the 3' untranslated region (3'-UTR) of the heat shock protein 70 (type I) gene (HSP70-I) have previously been used for typing of Leishmania but not with Leishmania (Mundinia) martiniquensis and L. (Mundinia) orientalis, newly identified human pathogens. Here, the 3'-UTRs of HSP70-I of L. martiniquensis, L. orientalis, and 10 other species were sequenced and analyzed. PCR-Restriction Fragment Length Polymorphism (RFLP) analysis targeting the 3'-UTR of HSP70-I was developed. Also, the detection limit of HSP70-I-3'-UTR PCR methods was compared with two other commonly used targets the 18S small subunit ribosomal RNA (SSU-rRNA) gene and the internal transcribed spacer 1 region of the rRNA (ITS1-rRNA) gene. Results showed that HSP70-I-3'-UTR PCR methods could be used to identify and differentiate between L. martiniquensis (480-2 bp) and L. orientalis (674 bp) and distinguished them from parasites of the subgenus Viannia and of the subgenus Leishmania. PCR-RFLP patterns of the 3'-UTR of HSP70-I fragments digested with BsuRI restriction enzyme successfully differentiated L. martiniquensis, L. orientalis, L. braziliensis, L. guyanensis = L. panamensis, L. mexicana = L. aethiopica = L. tropica, L. amazonensis, L. major, and L. donovani = L. infantum. For the detection limit, the HSP70-I-3'-UTR PCR method could detect the DNA of L. martiniquensis and L. orientalis at the same concentration, 1 pg/µL, at a similar level to the SSU-rRNA PCR. The PCR that amplified ITS1-rRNA was more sensitive (0.01 pg/µL) than that of the HSP70-I-3'-UTR PCR. However, the sizes of both SSU-rRNA and ITS1-rRNA PCR amplicons could not differentiate between L. martiniquensis and L. orientalis. This is the first report of using HSP70-I-3'-UTR PCR based methods to identify the parasites causing leishmaniasis in Thailand. Also, the BsuRI-PCR-RFLP method can be used for differentiating some species within other subgenera.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Leishmaniasis
/
Protozoan Proteins
/
Polymerase Chain Reaction
/
HSP70 Heat-Shock Proteins
/
3' Untranslated Regions
/
Molecular Typing
/
Leishmania
Type of study:
Diagnostic_studies
/
Evaluation_studies
Limits:
Humans
Country/Region as subject:
Asia
Language:
En
Journal:
PLoS Negl Trop Dis
Journal subject:
MEDICINA TROPICAL
Year:
2021
Document type:
Article
Affiliation country: