Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 79-80

REPORTE CORTO/SHORT REPORT

Presented in the Congress Biotecnologia Habana'94. La Habana, Cuba, Nov. 28 - Dec. 3, 1994

A NONRADIOACTIVE, BRANCHED DNA-BASED TECHNIQUE FOR DETECTION OF Trypanosoma brucei spp. IN BLOOD

Eva Harris^1, J. Kolberg^2, M. Urdea^2 and Nina Agabian^1

^1Intercampus Program in Molecular Parasitology, University of California at San Francisco, 333 California Avenue, Suite 150, San Francisco, CA, 94118; ^2Nucleic Acid Systems, Chiron Corporation, 4560 Horton Street, D-2 Emeryville, CA 94608- 2916.

Code Number: BA95020
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INTRODUCTION

Since their recent advent, molecular diagnostic methodologies have proven to be very useful and offer a number of advantages over microscopic, biochemical, and immunologic procedures for the detection of pathogens. To date, target amplification methods such as the polymerase chain reaction (PCR) have been the most commonly applied molecular techniques. The branched DNA (bDNA) Signal Amplification Assay is an alternative hybridization based system for the sensitive and rapid detection of agents of infectious disease. This molecular technique amplifies the signal from a target molecule rather than the target itself, and thus avoids artifactual problems that have hampered other molecular diagnostic methodologies (1).

We have developed a nonradioactive bDNA-based assay for detection of Trypanosoma brucei in clinical samples. T. brucei gambiense and T. brucei rhodesiense are the etiologic agents of sleeping sickness, and the disease is considered a major health problem in many African countries for humans as well as for cattle, which are infected with T. brucei. The accurate diagnosis of African sleeping sickness by direct blood examination is problematic due to the wave-like fluctuations in levels of parasites present in biological samples, while immunologic tests are hampered by the trademark antigenic variation of T. brucei used by the parasite to avoid the host immune response. We have developed a sensitive and specific diagnostic assay for African trypanosomiasis, regardless of the stage of infection, variable antigen type (VAT), or subspecies of T. brucei.

MATERIALS AND METHODS

In the branched DNA assay, crude lysates of samples are denatured and hybridized in solution to two sets of oligonucleotide probes (50 mers). One set serves to capture the target sequence from the organism of interest by hybridizing to both the target sequence and oligonucleotide probes bound to microtiter dish wells. The other set of probes contains regions complementary to both the target sequence and to branched DNA molecules and serves to amplify the signal. Each of the 45 branches in this amplifier structure then hybridizes to an alkaline phosphatase-labeled probe. Finally, the complex is detected by the addition of an enzyme-triggerable chemiluminescent substrate, and light emission is measuredwith a luminometer or a Polaroid camera (1). Plasma and Buffy coat were prepared from heparinized blood samples and used directly in the T. brucei bDNA assay (2).

RESULTS AND DISCUSSION

Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets, namely the 177 bp satellite repeat (3) and the RIME sequences (4, 5), and the appropriate oligonucleotide probes were designed and tested. Parasites were detected in biological samples with clinically relevant sensitivity (5-10 parasites/mL of blood), and comparable limits of detection were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes and bloodstream trypomastigotes. Analysis of the species specificity of the T. brucei bDNA assay with serial dilutions of purified DNAs revealed a strong signal from the three T. brucei subspecies and no signal from a variety of related organisms.

Thus, the branched DNA technology offers certain advantages over alternative molecular techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from cross-contamination artifacts.

REFERENCES

1. URDEA, M. S. (1993). Clinical Chemistry 39:725-726

2. HARRIS, E. et al. Manuscript in preparation.

3. SLOFF, P. et al. (1983). J. Molecular Biology 167:1-21

4. HASAN, G. et al. (1984). Cell 37:333- 341

5. KIMMEL, B. E. et al. (1987). Molecular and Cellular Biol. 7:1465-1475

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud