Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples.
Baylisascaris procyonis is a helminth parasite commonly found in North American raccoons (Procyon lotor) that is a cause of clinical neural, ocular, and visceral larva migrans in humans when infective eggs are ingested. Rapid detection of B. procyonis eggs in contaminated soil and water would assist public health analysts in evaluating risks associated with public exposure to areas of known raccoon activity. In this study, a molecular beacon probe-based real-time polymerase chain reaction (PCR) assay was developed to enable rapid and specific detection of eggs of Baylisascaris spp. The molecular beacon assay targeted the cytochrome oxidase subunit 2 (cox-2) gene of B. procyonis. To determine method sensitivity, experiments testing various egg levels (250, 25, and five eggs) were performed by seeding into 0.5-g soil samples or 0.5-mL water samples. Different soil sample types were extracted using a commercial nucleic acid extraction kit. Specificity testing using previously characterized helminth tissue specimens indicated that the assay was specific to Baylisascaris spp. Little real-time PCR inhibition was observed for most of the soil and water samples. A seed level of 250 eggs was detected for all soil types, and two seed levels (25 and five eggs) were detected for surface water samples. These results demonstrate that the reported real-time PCR assay was effective for the sensitive detection of B. procyonis in a wide range of soil types, and should be a useful tool for investigations of soil or water potentially contaminated with eggs of this parasite.
Gatcombe
Rachel R
RR
National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-36, Atlanta, GA 30341, USA.
Jothikumar
Narayanan
N
Dangoudoubiyam
Sriveny
S
Kazacos
Kevin R
KR
Hill
Vincent R
VR
D000818:Animals; D001199:Ascaridoidea / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000235:genetics; D005136:Eye Proteins; D015801:Helminth Proteins / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D014452:Ubiquitins; D014867:Water / Q000469:parasitology*
D015342:DNA Probes; D005136:Eye Proteins; D015801:Helminth Proteins; C429294:UBL5 protein, human; D014452:Ubiquitins; D014867:Water; D003576:Electron Transport Complex IV