title: Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR.
abstract: The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH(3)-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.
authors.affiliation: Department of Civil and Environmental Engineering, University of California, Berkeley, CA 94720-1710, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Raynal
authors.forename: Eric N
authors.initials: EN
authors.lastname: Villegas
authors.forename: Kara L
authors.initials: KL
authors.lastname: Nelson
mesh_terms: D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D017165:Ascaris suum / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology* / Q000528:radiation effects; D018509:DNA, Helminth / Q000032:analysis; D006358:Hot Temperature / Q000009:adverse effects; D006863:Hydrogen-Ion Concentration; D010063:Ovum / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D010270:Parasite Egg Count / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D018509:DNA, Helminth; D000641:Ammonia