When scientists used DNA Extraction Kit to find the results of contamination about DNA extraction reagents, they found out how easily the defects occur. Therefore filtration on the nucleic acid is considered an effective way of removing the reagent-derived contamination.
The membrane-sensitive bacterial DNA extractions and its relationship with absolute quantitation of recovery efficiencies
While doing the microbiological risk assessments, the researchers use various sophisticated tools like RNA Extraction Kit to quantify the waterborne pathogens.
During the process, it is mandatory to determine the DNA recovery to acquire the [revise efficiency. That is especially required before molecular measurements of the quantification.
During the study, the scientist had the DNA recovery efficiency and the quality depicted by five standard extraction methods. This also includes the two major phenol-chloroform extractions.There is also the use of mechanical shearings and the three commercial kits used for extractions.
The extraction of DNA from the indigenous bacteria mix culture is derived from river water. All of the methods regarding the experimentation were giving comparatively satisfying results. The results were primarily extracted from the pellet sample that was collected via the centrifugation.
The commercial used in the process provides shockingly low yields of DNA. The was then integrated by using enzymatic lysis, phenol extraction, bath sonication and the alcohol precipitation. A plasmid with human GAPDH gene fragment demonstrates an appropriate spiking to control the absolute DNA recovery efficiency. Therefore the unexpected low efficiency by any commercial kit implies the underestimation of pathogenic bacteria. This all should gain a fair concern in the domain of pathogen monitoring.
Are there intricacies regarding the assessment of human microbiome in epidemiologic studies?
In the previous decades, there was an assessment of a remarkable relationship found between the dysbiosis of human microbiota and the detrimental effects on the heath. This current review aims to signify the major pitfalls and challenges confronted during every stage of microbiota research. Right from the study design to the sample collection to sequencing and finally to nucleic acid extraction, there is statistical data analysis.
Although the majority of the studies have put their focus on assessing the internal composition in microbiota, there are not enough studies done on the causal roles of bacteria, viruses, archaea and fungi. That is interestingly shocking, because all play a significant role in influencing the disease states.
The microbiome research is a bit older and thus many aspects of it can be quite challenging. This also includes the complication and personalized aspects of microbial communities and the impact it has on exogenous confounding factors. Given the findings, the scientist deeply felt the urge to apply the basic or fundamental principles of epidemiology, ecology and the more advanced software tools. This is all essential in the rapidly evolving technological, evolving genomic and the analytical landscapes.
The increasingly incorporating human microbiome research in the huge epidemiologic studies reveal the intricate relationships that humans have with their microbial partners. This also helps to offer the interventional opportunities that are needed to improve human health by ten fold.
The errors in ribosomal sequence datasets and the usage of PCR-coupled ‘pan bacterial’
In order to identify and investigate the host-associated and environmental bacterial communities, the PCR-coupled sequencing is approached.
Some of these primers are also used for the amplification of eukaryotic DNA. This further leads to a submission of datasets that are mistakenly noted as prokaryotic sequences. Consequently, the present note is helpful at sending a message regarding the risk of submitting the incorrectly annotated sequence. This also suggests a reliable approach for the sequencing of 16S rRNA genes.
(Tamimount Mohammadi et al., 2004, #)
The DNA extraction of membrane-sensitive bacteria and the absolute quantitation of recovery efficiencies
If you seriously want to assess the microbiological risk assessment (MRA) in waterborne pathogens, then you would require the absolute quantification. DNA determination is a vital step for DNA recovery and its efficiency before the quantitative molecular measurements. Although this detail has a paramount importance, sadly it has faced major dejection.
The commercial kit reveals a very low yield of DNA for all the membrane filtered samples. That is mainly because of the DNA absorption on the surface of the membrane.
Shi, X. J., Zhang, M., Liu, G., Zhao, J., Li, H., Yang, Z., Bai, H., Liang, P., & Lu, Y. (2020). Membrane-sensitive bacterial DNA extractions and absolute quantitation of recovery efficiencies,. Science of The Total Environment,. https://www.sciencedirect.com/science/article/abs/pii/S0048969719351174
Tamimount Mohammadi, Reesink, H. W., M.J.E., C., Vandenbroucke -Grauls, & Paul H.M. (2004, 11 18). Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. Journal of Microbiological Methods, 61(2), 285-288. https://www.sciencedirect.com/science/article/abs/pii/S0167701204003392