title: A real-time PCR method for quantifying viable ascaris eggs using the first internally transcribed spacer region of ribosomal DNA.
abstract: Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.
authors.affiliation: Department of Civil and Environmental Engineering, MS 1710, University of California, Berkeley, CA 94720-1710, USA.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Pecson
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Barrios
authors.forename: David R
authors.initials: DR
authors.lastname: Johnson
authors.forename: Kara L
authors.initials: KL
authors.lastname: Nelson
mesh_terms: D000818:Animals; D017165:Ascaris suum / Q000254:growth & development* / Q000302:isolation & purification; D017931:DNA Primers; D018509:DNA, Helminth / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D010063:Ovum / Q000254:growth & development*; D010270:Parasite Egg Count; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D018510:RNA, Helminth / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D018510:RNA, Helminth; D012335:RNA, Ribosomal