Table of Contents
Introduction
In today’s society, DNA isolation is known to be a low-cost and simple testing method that involves the purification of DNA through a series of physical and chemical experiments. In this study, five commercial kits for DNA isolation were tested in order to compare which kit is more efficient at extracting DNA from blood from individuals of four microbial groups that contained symptoms of sepsis. The four microbial groups tested in this study are yeast: Candida albicans, filamentous fungus: Aspergillus fumigatus, Gram negative: Escherichia coli, and Gram positive: Staphylococcus aureus. These bacteria and fungus are varied by their cell wall structure (Gosiewski et al., 2013).
Sepsis is a disease that involves the body sending an overdose of chemicals and antibodies to fight off an infection, which results in inflammation in multiple organs and may be fatal. This infection typically occurs when some type of bacteria or fungus enters the bloodstream. The diagnosis of factors causing infections is complicated but important in treating these blood infections (Westh et al., 2009).
The samples in this experiment were prepared for DNA isolation by mechanical disruption, chemical lysis, and thermal lysis (Gosiewski et al., 2013). Lysis is described as the rupture of a cell’s plasma membrane by disintegration (Mason et al., 2016). After the samples are prepared, the five commercial kits; GeneMATRIX Quick Blood DNA Purification Kit (EURx), GeneJET (Fermentas), QIAamp DNA Blood (QIAGEN), Blood Mini (A&A Biotechnology), and Genomic Mini (A&A Biotechnology) are used for the actual extraction of DNA from the samples.
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The use of real time polymerase chain reaction (rtPCR) method then allows for the amplification of DNA for all processes and determination of microbial DNA in the blood (Gosiewski et al., 2013). PCR imitates DNA replication through the repetition of denaturation, annealing of primers, and synthesis (all in that order). Currently, PCR is made faster and requires less labor than when it was initially discovered due to a thermostable DNA polymerase identified as Taq polymerase. The invention of machines with heating blocks that can be cycled over big temperature ranges also allows for a more accurate and rapid process (Mason et al., 2016).
The advantages of this method of DNA isolation is that these tests are simple and affordable to perform. However, the disadvantages include time consumption and difficulty in detecting microbes in blood. Nucleic acid detection is used in this study to enable a higher detection rate than normal. The main objective of this experiment was to detect which method of DNA isolation is best suited for detecting bacteria and fungi DNA in human blood and to also discover which commercial kit is most efficient for DNA isolation (Gosiewski et al., 2013).
Recent Research
When this experiment was conducted, the scientists performing the lab were aware that many other scientists have performed similar experiments and have recommended to isolate either bacterial or fungal DNA from blood, but not both. However, these other experiments were conducted with different volumes of blood and commercial kits, making it difficult to compare these results. To develop an efficient method for isolating both bacterial and fungal DNA from blood, several methods are applied in attempt to obtain a high quality DNA matrix. In this case, sepsis is the microbial disease being experimented because there is still the demand for more diagnostic information on this infection (Sugita et al., 2012).
Before DNA isolation is performed, there are a few simple steps that must be done to prepare the samples. The samples of blood containing the microbial DNA all underwent erythrocyte lysis in 0.17 M ammonium chloride solution. Then, glass beads were used to perform mechanical disruption, a way to lyse cells, in a FastPrep machine. Afterwards, enzymes were used to perform enzymatic lysis of bacteria at 37° Celsius. The enzymes involved in this process are called lyticase 40 U, lysostaphin, and lysozyme. The purpose of these enzymes are to digest the cell walls of these microbes, so we may gain access to the nucleic acids.
Before the PCR method can be used, the microbes’ DNA must be isolated first so we may be able to detect the amount of microbes in the blood. The commercial kits that were listed are then used so DNA is isolated from the prepared samples. 100 Microliters of Tris buffer is used for elution of DNA from the columns (Gosiewski et al., 2013). The purpose of buffers in labs are to maintain a desired range of pH of a sample or solution. Tris buffer is commonly used because of its ability to produce a neutral range of pH (Mason et al., 2016). DNA was extracted from a total of sixteen samples for each set.
Now that the DNA has been isolated, a thermocycler (also known as a thermo cycler) CFX96 (BioRad) is used to apply real-time polymerase chain reaction (rtPCR) to amplify the DNA. Species-specific primers are involved for DNA synthesis that flank the area to be replicated and taqman probes are essential for the specificity of PCR. Sequences of oligonucleotides, or short DNA molecules, are utilized for the amplification procedures of each of the four microbes (Gosiewski et al., 2013). The first step of the PCR machine is to denature the DNA by heating the samples to 95° Celsius for about 2 minutes. Next, the DNA is cooled to 61° Celsius for about half a minute in order to allow the primers to anneal to DNA. Afterwards, the DNA is reheated to 72° Celsius, the optimal temperature for Taq DNA polymerase to extend the primers (Mason et al., 2016). This process of heating, cooling, and reheating is cycled 50 times for each microbial to completely undergo PCR.
After each sample has been processed through rtPCR, a spectrophotometer is utilized to determine the concentration and purity of DNA isolated in the samples at wavelengths of 260 absorbance and 280 absorbance. A Nanodrop machine is then utilized for the measurement of heme concentration at a wavelength of 388 absorbance in the DNA extracts produced from blood (Lombardo 2015). The PCR method detection limit is known as the minimal number of microbes identified, which may be detected through the blood meanwhile the rtPCR is taking place. The amplification detection limit is significant in this experiment because it defines the relationship between CT values, the required number of cycles, and the baseline established at 30 RFU. The detection limit was established for volumes of 1, 1.5, and 5 mL of blood. In correspondence to these volumes, DNA was eluted from these columns through utilization of a specific amount of elution buffer for each blood sample volume.
The scientists that conducted this experiment aimed at addressing which commercial kit is most effective in comparison to others when attempting to extract DNA from all four microbes aimed at being detected in blood. As a result, the GeneMatrix Quick Blood DNA Purification Kit (EURx) proved to be significantly better than the others in comparison to concentration and purity values. Another reason this commercial was more significant than the others was because it allowed the DNA amplification signal to be produced more quickly than the others could.
This answer was produced through careful examination of purity and concentration of all the microbial DNA extracted from blood after rtPCR was conducted and the method detection limit was evaluated. From this data, the scientists were also able to determine the detection limit of the fungi to be lower, meanwhile both species of bacteria involved had identical values. The scientists believe this was due to the fact that lysis ruptures the cell wall of bacteria more easy than it could for fungus, allowing for the extraction of more DNA from the same amount of cells that the fungus produced (Gosiewski et al., 2013).
Conclusion
The significance of this experiment is the search for an efficient way of being able to detect bacteria and fungus DNA in human blood. Diagnosis of diseases such as sepsis, where bacteria or fungus enters the bloodstream, are hard to identify and are in need of new methods to be able to distinguish them. As of today, blood culture still remains the ideal method of diagnosis because its readings are more accurate and easier to perform. Although there is a better method for this experiment, it is important to question whether it is possible for a more efficient way of detecting the amount of fungal or bacterial DNA extracted from blood samples. In this study, the scientists evaluate this amount through the detection limit, which only accounts for about 20% of the culture to identify the growth of microbes in the samples.
In other experiments involving PCR, DNA quantification is determined by utilizing fluorescent DNA-binding dyes that fluoresce when illuminated by a laser in the PCR machine. After each cycle in the PCR, the reaction irradiates and detectors are able to quantify what is being illuminated. This method is useful for many reasons, especially because the quantity of DNA is increased through each PCR cycle. This also increases the quantity of fluorescence since more dye binds to the DNA. If fluorescent DNA-binding dyes were used in this experiment, then the amount of fungal and bacterial DNA would be easier to detect from human blood samples (Mason et al., 2016). It is important to discover a new way of detecting microbial DNA in human blood since this process leads to infection and even death in some cases.
Since this experiment was limited to only 4 different microbes and 5 separate commercial kits, it is important to test other microbes and commercial kits in order to get a more broad analysis. If more bacteria and fungus are tested using more commercial kits, then we will gain a larger comparison and understanding of how to effectively identify and extract microbial DNA from human blood. It is very important to discover this because, again, these types of infections can be life threatening. As of today, there is no known better method for the diagnosis of these infections besides blood culture (Gosiewski et al., 2013).
References
- Gosiewski, T., Szała, L., Pietrzyk, A., Brzychczy-Włoch, M., Heczko, P. B., & Bulanda, M. (2013). Comparison of methods for isolation of bacterial and fungal DNA from human blood. Current microbiology, 68(2), 149-55.
- Lombardo ME, Araujo LS, Ciccarelli AB, Batlle A. A spectrophotometric method for estimating hemin in biological systems. Anal Biochem. 2015;341:199–203.
- Mason, Kenneth A., Losos, Jonathan B., Singer, Susan R., et al. “Chapter 17: Biotechnology.” Biology 11th Edition, by Peter H. Raven and George B. Johnson, McGraw-Hill Education, 2016, pp. 327–349.
- Sugita S, Kamoi K, Ogawa M, Watanabe K, Shimizu N, Mochizuki M. Detection of Candida and Aspergillus species DNA using broad-range real-time PCR for fungal endophthalmitis. Graefes Arch Clin Exp Ophthalmol. 2012;250:391–398.
- Westh H, Lisby G, Breysse F, Böddinghaus B, Chomarat M, Gant V, Goglio A, Raglio A, Schuster H, Stuber F, Wissing H, Hoeft A. Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis. Clin Microbiol Infect. 2009;15:544–551.
