Conclusion and future work
In conclusion, intact and adequate DNA obtained during extraction testifies to the effectiveness and efficiency of the method employed. During extraction of DNA from samples, there are several factors that can lead to sheared, impure, and/or inadequate samples of DNA including physical strain such as vortexing. Pipetting, presence of impurities such as RNA as well as the fragile nature of DNA strands especially those less than 150 kb can affect the quality and quantity of DNA extracted. During the research, potassium ethyl xanthogenate (XS) buffer provided the best quality 20 kbp when assayed through agarose gel electrophoresis. XS buffer was the optimal method for DNA extraction method as the strands were additionally distinct and had detectable specific biomarkers compared to other methods. While methods such as Chelex+RNase produced the highest concentration of DNA (1501.5 hg/µl), the DNA extracts not only had RNAs impurities; they were severely damaged through shearing.
The singleplex and multiplex PCR experiments were successful employed during the experiments as they determined which DNA extraction was optimal by identifying the distinctiveness of the bands on strands from DNA samples extracted by each method. The experiments also helped in identifying which method produced DNA samples with specific biomarkers. The experiments were thus important in choosing which method was to be used for DNA extraction in this study. Therefore, this study presented a significant assay for pathogenic E. coli detection in lake water and other recreational considering that nowadays few studies examine the potential pathogens in such waters.
Recommendations for future research
As shown in the Pathogenicity Table, it is possible to establish statistical significance for these results. Such studies will contribute to the further understanding of pathogenicity of recreational waters depending on factors that are unique to certain areas. Future studies can also expand and include other water bodies including sources of water for domestic use especially those that are susceptible to contamination by pathogens. Quantifying pathogenicity will offer a great insight that can help in fields beyond science including legal matters especially when it comes to compensation.
In future, based on this study, I recommend that more tests and comparative studies should be conducted on UPEC virulence genes because Uropathogenic E. coli (UPEC) has many virulence genes (VGs). When these VGs are compared to those, found in other sources, it would be possible to determine with greater accuracy whether the water is contaminated with the VGs found in UPEC.
Running the gel electrophoresis to detect samples after PCR is not only time consuming but can also lead to shearing and eventual damage of DNA samples extracted. This significantly hampers DNA extraction and identification while is also time consuming. Therefore, I recommend that future research should also focus on developing a multiplex-qPCR that would help avoiding such scenarios. I also recommend that a colony hybridization using a probe be made to allow for more effective and effective studies in virulence genes.
During the study, it was established that Chelex+RNase and Chelex produced the highest quantities of DNA. However, the extracts were contaminated and sheared hence lacked distinct bands and biomarkers. Future research should focus on how such methods can be made effective by eliminating factors that lead to contamination and shearing of DNA. This will ensure that an effective method is put in place that allows for extraction of large amounts of DNA, which are very intact and can be easily identified using the specific identifiable markers.