Start to Finish Detection of Cryptosporidium for in Field Use on Water Samples via Lauroylsarcosine sodium Salt (LSS) Extraction, Loop Mediated Isothermal Amplification (LAMP), and DNA Precipitation
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Today, many diseases that are rare in developed nations are still prevalent in other countries due to limited technological access and resources. Among these diseases is Cryptosporidiosis, a common gastro-intestinal infection with currently no known treatments. Cryptosporidiosis is caused by the parasite Cryptosporidium. This parasite spreads through the environment as an oocyst, causing self-limiting diarrhea in healthy humans. These symptoms are often more dangerous in immuno-compromised individuals and children, whose immune systems are still developing. The rugged Cryptosporidium oocysts are able to survive treatment with most disinfectants. Thus, to prevent outbreaks, there must be early detection, followed by rapid treatment of the water supply and/or limited exposure to infected water supplies. Current methods of parasite testing for water samples are limited in their function by cost, access to materials, and ability to run equipment. Improvements in cost-effective, easy to use alternatives to traditional techniques, such as the use of lauroylsarcosine sodium salt (LSS) for DNA extraction and the loop mediated isothermal amplification (LAMP) method of amplification, will facilitate the detection of Cryptosporidium in the field and in impoverish nations.In this experiment, a five primer LAMP reaction was used in combination with the LSS reaction to eliminate otherwise essential electrical equipment in the detection process, expediting and easing in-field detection. The LSS was found to decrease the efficiency of the LAMP reaction, but its effects were mediated by changing the buffer used in the LAMP reaction. Amplicons were visualized using gel electrophoresis in this experiment. However, methods of detection were tested for incorporation into the test. These techniques will lead to the future of cost efficient detection of Cryptosporidium in water samples.
Cryptosporidium; Loop-Mediated Isothermal Amplification; parasite detection; cost-efficiency; optimization
dissertation or thesis