title: Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi.
abstract: Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1-10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes.
authors.affiliation: Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR Intertryp, F-34398 Montpellier, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Desquesnes
authors.forename: Desquesnes
authors.initials: D
authors.lastname: Marc
authors.forename: Ketsarin
authors.initials: K
authors.lastname: Kamyingkird
authors.forename: Kamyingkird
authors.initials: K
authors.lastname: Ketsarin
authors.forename: Sarawut
authors.initials: S
authors.lastname: Yangtara
authors.forename: Yangtara
authors.initials: Y
authors.lastname: Sarawut
authors.forename: Cristina
authors.initials: C
authors.lastname: Milocco
authors.forename: Milocco
authors.initials: M
authors.lastname: Cristina
authors.forename: Sophie
authors.initials: S
authors.lastname: Ravel
authors.forename: Ravel
authors.initials: R
authors.lastname: Sophie
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Wang
authors.forename: Wang
authors.initials: W
authors.lastname: Ming-Hui
authors.forename: Zhao-Rong
authors.initials: ZR
authors.lastname: Lun
authors.forename: Lun
authors.initials: L
authors.lastname: Zhao-Rong
authors.forename: Serge
authors.initials: S
authors.lastname: Morand
authors.forename: Morand
authors.initials: M
authors.lastname: Serge
authors.forename: Sathaporn
authors.initials: S
authors.lastname: Jittapalapong
authors.forename: Jittapalapong
authors.initials: J
authors.lastname: Sathaporn
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D012376:Rodent Diseases / Q000469:parasitology*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014350:Trypanosoma lewisi / Q000235:genetics* / Q000302:isolation & purification; D014352:Trypanosomiasis / Q000469:parasitology / Q000662:veterinary*
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer