Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: diagnosis of chronic Chagas' disease

https://doi.org/10.1016/0166-6851(91)90116-NGet rights and content

Abstract

A 6 M guanidine-HCI/0.2 M EDTA solution was used to lyse and store whole blood specimens. DNA stored in guanidine-EDTA-blood (GEB) lysate was found to be undergraded after incubation at 37°C for 1 month, suggesting that this represents an appropriate reagent for transport of blood samples from the field to a laboratory for analysis. Trypanosoma cruzi kinetoplast DNA in GEB lysate can be cleaved using the chemical nuclease, 1,10-phenanthroline-copper ion (OP-Cu2+). This procedure liberates linearized minicircle molecules from network catenation, distributing them through out the lysate, and allowing a small aliquot of the original lysate to be analyzed by PCR amplication. This increases the sensitivity of the method dramatically for the detection of small numbers of trypanosomes in a large volume of blood. DNAs isolated from aliquots of T. cruzi-positive GEB lysates were polymerase chain reaction (PCR)-amplified with 3 sets of T. cruzi-specific kDNA minicircle primers, yielding the 83-bp and 122-bp conserved region fragments and the 330-bp variable region fragments. The PCR products were analyzed by gel electrophoresis and/or hybridization. Results indicate that a single T. cruzi cell in 20 ml of blood can be detected by this method. Blood samples from several chronic chagasic patients were tested. Amplification of T. cruzi kDNA minicircle sequences was obtained in a1 cases, even when xenodiagnosis was negative. This PCR-based test should prove useful as a replacement or complement for xenodiagnosis or serology in clinical and epidemiological studies of chronic Chagas' disease.

References (29)

  • G.H. Keller et al.

    Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization

    J. Clin. Microbiol.

    (1990)
  • D. Shibata et al.

    Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus

    J. Infect. Dis.

    (1988)
  • J.W. Zolg et al.

    High salt lysates: A simple method to store blood samples without refrigeration for subsequent use with DNA probes

    Am. J. Trop. Med. Hyg.

    (1988)
  • D.F. Wirth et al.

    Leishmaniasis and malaria: DNA probes for diagnosis and epidemiologic analysis

    Ann. NY Acad. Sci.

    (1989)
  • Cited by (0)

    View full text