Quantification of PCR amplification products of Brugia HhaI repeat DNA using a semiautomated Q-PCR system.
The sensitive, rapid and species-specific diagnosis of Brugia infections in humans or animal models is important in determining the level of parasitemia and the efficacy of chemotherapy or vaccinations. The HhaI family of highly repeated DNA sequences from Brugia have been useful in polymerase chain reaction (PCR)-based diagnosis of brugian filarial infections in blood samples and in mosquitoes. A PCR assay was developed using a biotinylated primer, a non-biotinylated primer and a species-specific chemiluminescent probe [tris[2,2'bipyridine] ruthenium (II) chelate, TBR] to detect PCR amplified Hhal family repeats. Individual blood samples from jirds infected with Brugia malayi or B. pahangi and with different levels of microfilaremia were tested in this assay. Known concentrations of Brugia DNA and DNA from the blood of uninfected control jirds were used as positive and negative controls, respectively. The PCR products generated by this method were analyzed using a semi-automated quantitative (Q)-PCR system. The levels of parasite DNA can be calculated from the luminosity units generated. Significant amounts of parasite DNA were detected in blood samples from infected jirds, and these values were correlated with the levels of microfilaremia. In contrast, reductions in circulating microfilaria following treatment with ivermectin correlated with low levels of measurable DNA. Using this system, we were also able to detect HhaI repeat DNA in the spleens of B. pahangi- infected jirds at 56 days post-infection when circulating microfilariae were not readily detectable. The results indicate that the species-specific Hhal Q-PCR detection and quantification method is rapid and sensitive, is useful in the detection of Brugia DNA in blood and other tissues and is suited for use in clinical settings because it does not require radioactive isotopes and gel-based protocols.
Rao
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Department of Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA. rrao@imgate.wustl.edu
Williams
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Klei
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D000818:Animals; D001331:Automation; D002009:Brugia / Q000235:genetics / Q000302:isolation & purification*; D016207:Cytokines / Q000235:genetics; D017931:DNA Primers; D018509:DNA, Helminth / Q000032:analysis*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000032:analysis / Q000235:genetics*; D004605:Elephantiasis, Filarial / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*; D005369:Filaricides / Q000494:pharmacology; D007559:Ivermectin / Q000494:pharmacology; D008163:Luminescent Measurements; D008198:Lymph Nodes / Q000469:parasitology; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D013154:Spleen / Q000469:parasitology
D016207:Cytokines; D017931:DNA Primers; D018509:DNA, Helminth; D005369:Filaricides; D007559:Ivermectin; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases