Characterization of a candidate Trypanosoma rangeli small nucleolar RNA gene and its application in a PCR-based parasite detection.
In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.
Morales
Liliana
L
Laboratorio de Parasitología Molecular, Departamento de Microbiología, Facultad Ciencias, Universidad Javeriana, Carrera 7 No 43-82, Lab. 113, Bogotá, Colombia.
Romero
Ibeth
I
Diez
Hugo
H
Del Portillo
Patricia
P
Montilla
Marleny
M
Nicholls
Santiago
S
Puerta
Concepción
C
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D014355:Chagas Disease / Q000175:diagnosis* / Q000469:parasitology; D017931:DNA Primers; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification*; D003937:Diagnosis, Differential; D015723:Gene Library; D007303:Insect Vectors / Q000469:parasitology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D016053:RNA, Protozoan / Q000235:genetics; D020537:RNA, Small Nucleolar / Q000235:genetics*; D012239:Rhodnius / Q000469:parasitology; D016415:Sequence Alignment; D013045:Species Specificity; D014345:Trypanosoma / Q000235:genetics* / Q000302:isolation & purification; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D020537:RNA, Small Nucleolar