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Multiplex PCR on single unembryonated Ascaris (roundworm) eggs
Josefine Carlsgart1, Allan Roepstorff, Peter Nejsum
1Danish Centre for Experimental Parasitology, Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Dyrlaegevej 100, 1870 Frederiksberg C, Copenhagen, Denmark.
Abstract:
A sensitive and inexpensive method for DNA isolation and amplification by polymerase chain reaction (PCR) from single unembryonated Ascaris sp. eggs is described. The resistant shell of single eggs was crushed mechanically and PCR applied to the crude egg contents without any further purification steps. The ITS1 region of the rDNA and three regions of the mtDNA could be successfully amplified. Using two primer sets, it was possible to amplify the rDNA and mtDNA simultaneously in one single reaction. The ability to perform PCR on single unembryonated eggs may result in better and more precise species identification of eggs recovered from faecal material, environmental samples and possibly archaeological samples. In addition, single egg PCR makes it possible to perform population genetic studies without having to recover adult worms by deworming or autopsy.
Insights
We developed a simple method to amplify DNA from single Ascaris sp. eggs using polymerase chain reaction (PCR). This technique allows for precise species identification and genetic studies without needing adult worms.
Area of Science:
- Parasitology
- Molecular Biology
- Genetics
Background:
- Accurate identification of Ascaris species is crucial for public health and veterinary diagnostics.
- Current methods for DNA analysis often require multiple adult worms or extensive sample processing.
Purpose of the Study:
- To develop a sensitive, inexpensive method for DNA isolation and amplification from single unembryonated Ascaris sp. eggs.
- To enable precise species identification and population genetic studies using minimal starting material.
Main Methods:
- Mechanical crushing of single unembryonated Ascaris sp. eggs to release contents.
- Direct application of polymerase chain reaction (PCR) to crude egg contents without purification.
- Amplification of the ITS1 region of rDNA and three regions of mtDNA using specific primer sets.
- Simultaneous amplification of both rDNA and mtDNA in a single PCR reaction.
Main Results:
- Successful DNA amplification from single unembryonated Ascaris sp. eggs.
- Amplification of both nuclear (rDNA ITS1) and mitochondrial (mtDNA) DNA regions.
- Demonstration of simultaneous amplification of rDNA and mtDNA from a single egg.
- The method proved sensitive and inexpensive.
Conclusions:
- This single-egg PCR technique offers a highly precise method for Ascaris species identification in various sample types (fecal, environmental, archaeological).
- It facilitates population genetic studies without the need to collect adult worms, simplifying research and diagnostics.
