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Applied and Environmental Microbiology, December 2006, p. 7864-7872, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01983-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA{triangledown}

Brian M. Pecson,1 José Antonio Barrios,2 David R. Johnson,1 and Kara L. Nelson1*

Department of Civil and Environmental Engineering, MS 1710, University of California, Berkeley, California 94720-1710,1 Instituto de Ingeniería UNAM, Apartado Postal 70-402, Ciudad Universitaria, 04510 Coyoacan, Mexico2

Received 21 August 2006/ Accepted 11 October 2006

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.

* Corresponding author. Mailing address: Department of Civil and Environmental Engineering, MS 1710, University of California, Berkeley, CA 94720-1710. Phone: (510) 643-5023. Fax: (510) 642-7483. E-mail: nelson{at}ce.berkeley.edu.

{triangledown} Published ahead of print on 20 October 2006.

Applied and Environmental Microbiology, December 2006, p. 7864-7872, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01983-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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