A real-time PCR method for quantifying viable ascaris eggs using the first internally transcribed spacer region of ribosomal DNA

Appl Environ Microbiol. 2006 Dec;72(12):7864-72. doi: 10.1128/AEM.01983-06. Epub 2006 Oct 20.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ascaris suum / growth & development*
  • Ascaris suum / isolation & purification
  • DNA Primers
  • DNA, Helminth / analysis
  • DNA, Ribosomal Spacer / analysis*
  • Ovum / growth & development*
  • Parasite Egg Count*
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • RNA, Helminth / genetics
  • RNA, Ribosomal / genetics


  • DNA Primers
  • DNA, Helminth
  • DNA, Ribosomal Spacer
  • RNA, Helminth
  • RNA, Ribosomal