Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances.
Although there have been numerous microbial examinations of soil for the presence of human pathogenic developmental parasite stages of Ancylostoma caninum and Toxocara canis, molecular techniques (e.g. DNA extraction, purification and subsequent PCR) have scarcely been applied. Here, DNA preparations of soil samples artificially contaminated with genomic DNA or parasite eggs were examined by PCR. A. caninum and T. canis-specific primers based on the ITS-2 sequence were used for amplification. After the sheer DNA preparation a high content of PCR-interfering substances was still detectable. Subsequently, two different inhibitors of PCR-interfering agents (GeneReleaser, Bioventures Inc. and Maximator, Connex GmbH) were compared in PCR. Both substances increased PCR sensitivity greatly. However, comparison of the increase in sensitivity achieved with the two compounds demonstrated the superiority of Maximator, which enhanced sensitivity to the point of permitting positive detection of a single A. caninum egg and three T. canis eggs in a soil sample. This degree of sensitivity could not be achieved with GeneReleaser for either parasite Furthermore, Maximator not only increased sensitivity; it also cost less, required less time and had a lower risk of contamination. Future applications of molecular methods in epidemiological examinations of soil samples are discussed/elaborated.
Krämer
F
F
Institute of Parasitology, Hannover School of Veterinary Medicine, Bünteweg 17, D-30559 Hannover, Germany.
Vollrath
T
T
Schnieder
T
T
Epe
C
C
D000722:Ancylostoma / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D018509:DNA, Helminth / Q000032:analysis* / Q000097:blood / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012987:Soil / Q000469:parasitology*; D017166:Toxocara canis / Q000235:genetics / Q000302:isolation & purification*
D018509:DNA, Helminth; D012987:Soil