Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study.
To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500 microliters of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500 microliters of normal mice blood. Positive control: 500 microliters of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers: S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500 microliters of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.
Campos
Rozália F
RF
Laboratório de Doença de Chagas Experimental do Centro de Pesquisas Gonçalo Moniz da Fundação Instituto Oswaldo Cruz, Salvador, BA, Brasil.
Magalhães
Juracy B
JB
Reis
Eliana A G
EA
Reis
Mitermayer G
MG
Andrade
Sonia G
SG
D000818:Animals; D018105:DNA, Kinetoplast / Q000097:blood; D051379:Mice; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000302:isolation & purification*
D018105:DNA, Kinetoplast