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Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR
Citation:
Raynal, M., E. VILLEGAS, AND K. L. Nelson. Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR. JOURNAL OF WATER AND HEALTH. IWA Publishing, London, Uk, 10(4):594-604, (2012).
Impact/Purpose:
The objectives of this task are to: • Optimize and evaluate methods to isolate and detect viruses in biosolids • Evaluate a new method for detecting and determining the viability of Ascaris ova in a variety of sludge types. The research in this task will support LTG 4 of the Water Quality Multiyear Plan and the methods developed will be used for the occurrence studies recommended by the National Research Council report.
Description:
Aims: The goal of the study was to further develop an incubation-qPCR method for quantifying viable Ascaris eggs. The specific objectives were to characterize the detection limit and number of template copies per egg, determine the specificity of the method, and test the method with viable and inactivated larvated eggs. Methods and Results: 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 then tested against a panel of bacteria, fungi, protozoa, and helminths. No significant amplification was found with non-target DNA. Finally, larvated eggs were inactivated by four different treatments: 254-nm ultraviolet light, 2000 ppm NH3-N at pH 9, moderate heat (48 °C), and high heat (70 °C). Concentrations of treated eggs were then measured by direct microscopy and incubation-qPCR. Results revealed a decrease in qPCR signal following all four treatments and were in general agreement with the decrease in viable eggs determined by microscopy. Conclusions: The incubation-qPCR method for measuring the number of viable Ascaris eggs is effective and should be further developed. Significance and Impact of Study: An enumeration method for viable Ascaris eggs that produces results faster than direct microscopy provides a more practical and rapid approach for assessing biosolids.
