Species specific detection of Trypanosoma cruzi and Trypanosoma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA.
Several groups have recently developed molecular tests for the detection of Trypanosoma cruzi, the causative agent of Chagas' disease. Polymerase chain reaction (PCR) amplification of kinetoplast minicircle DNA sequences appears to be the most sensitive method. However, the specificity of PCR-based diagnostic methods was challenged when the complete sequence of Trypanosoma rangeli DNA minicircles was discovered. In the present study, we conducted. PCR experiments using the S35/S36 primers in Rhodnius prolixus and Balb/c mice with single and mixed infections of T. cruzi and/or T. rangeli. In single infections, the profile of each trypanosome was easily distinguishable in haemolymph, salivary gland and intestinal tissues and faeces of insect vectors. In mixed infections of anterior intestine (where T. rangeli is more predominant than T. cruzi), the DNA amplification profile of both parasites was observed simultaneously. Conversely, only the T. cruzi profile was observed in rectal ampulla (where T. cruzi is more abundant than T. rangeli). In mice with single infections of T. cruzi or T. rangeli, the profiles of amplified DNA were easily distinguishable in each case. The T. cruzi profile was dominant in most mixed infections, probably due to the fact that T. cruzi minicircles are more abundant and consequently compete more eagerly for annealing with the S35/S36 primers. In cases of mixed infections where T. rangeli was initially more abundant than T. cruzi, the specific T. rangeli 760 bp band was present for 7 days after infection and then this band and others ranging from 300 to 450 bp disappeared and only the typical T. cruzi 330 bp band remained. The S35/S36 primers used in polyacrylamide gel electrophoresis (PAGE) detected T. cruzi specifically, and prevented misdiagnosis due to the presence of T. rangeli. This technique can also be used to identify parasites in different stages of the infection (acute or chronic) in vertebrate hosts and to localize the parasites in the insect vector.
Vallejo
G A
GA
Departamento de Biología, Instituto de Ciencias, Universidad del Tolima, Ibagué, Colombia.
Guhl
F
F
Chiari
E
E
Macedo
A M
AM
D000818:Animals; D001483:Base Sequence; D014355:Chagas Disease / Q000175:diagnosis* / Q000469:parasitology; D016384:Consensus Sequence; D018105:DNA, Kinetoplast / Q000032:analysis* / Q000302:isolation & purification; D007303:Insect Vectors / Q000469:parasitology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012239:Rhodnius / Q000469:parasitology*; D013045:Species Specificity; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification; D014352:Trypanosomiasis / Q000175:diagnosis* / Q000469:parasitology
D018105:DNA, Kinetoplast