Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations.
Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.
Vargas
N
N
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26.077, CEP 05513-970 São Paulo, Brazil.
Souto
R P
RP
Carranza
J C
JC
Vallejo
G A
GA
Zingales
B
B
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D014355:Chagas Disease / Q000175:diagnosis / Q000453:epidemiology* / Q000469:parasitology; D003105:Colombia / Q000453:epidemiology; D004262:DNA Restriction Enzymes; D018105:DNA, Kinetoplast / Q000737:chemistry / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000737:chemistry* / Q000302:isolation & purification; D003937:Diagnosis, Differential; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012239:Rhodnius / Q000469:parasitology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*
D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D012335:RNA, Ribosomal; D004262:DNA Restriction Enzymes