Trypanosoma cruzi: optimization of polymerase chain reaction for detection in human blood.
We have optimized the conditions for DNA extraction and polymerase chain reaction (PCR) amplification to diagnose the presence of Trypanosoma cruzi DNA in the blood and serum of patients with chronic Chagas disease. The approximately 330-bp fragment of the kinetoplast minicircles was used as a target for amplification. The use of chemiluminescence on slot blots with a specific alkaline phosphatase-conjugated oligonucleotide probe detected specific product from as little as 0.1 fg of T. cruzi kDNA. An additional product of approximately 200 bp inadvertently amplified from the human genome was observed in human blood from T. cruzi-negative and -positive samples and served as an internal control of the amplification. Samples from other mammalian hosts were also assayed using the PCR protocol. The higher sensitivity of our PCR method observed in both acute and chronic phases of T.cruzi infections in mice and dog, respectively, could be useful in monitoring the course of infection during experimental drug tests in laboratory animals. Since this procedure showed a higher sensitivity than other protocols in the literature, it may be a suitable routine test in diagnosing Chagas disease, especially for patients presenting very low parasitemia levels.
Gomes
M L
ML
Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Macedo
A M
AM
Vago
A R
AR
Pena
S D
SD
Galvão
L M
LM
Chiari
E
E
D000818:Animals; D001483:Base Sequence; D014355:Chagas Disease / Q000097:blood / Q000469:parasitology*; D002908:Chronic Disease; D018105:DNA, Kinetoplast / Q000097:blood* / Q000737:chemistry; D004285:Dogs; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000097:blood / Q000469:parasitology*; D016133:Polymerase Chain Reaction; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D018105:DNA, Kinetoplast