DNA-Based Identification Methods For Campylobacter

Campylobacter: Infection, Diseases, Transmission

Disucuss about the DNA-based Identification Methods for Campylobacter species.

DNA-based identification methods are the best technique to detect a bacterium. Campylobacter species are Grams-Negative genus which takes the shape of the letter (s) and moves about smoothly (Castaño-Rodríguez, Kaakoush, Lee, & Mitchell, 2015). The species infects both animals and humans with diseases. However, the species majorly occupied the organ systems of the poultry animals. In many cases, the transmission of diseases by the bacterium occurs when an individual eats a poultry product that has the bacterium.  Additionally, human beings can be infected when they touch a poultry animal that has the bacterium. Campylobacter is large genus with numerous types of species. The Coli and Jejuni are the species which are known all over the scientific world (Castaño-Rodríguez et al., 2015).

Campylobacter jejuni is the causative agent of food-related infections (Nachamkin, & Nguyen, 2017). Once an individual has acquired the species, it can easily relocate the individual’s bloodstream. A person who has HIV/AIDS virus can infect another person with the virus plus the bacterium (Nachamkin, & Nguyen, 2017). The lari species affects children with continuously constant diarrhea (Nachamkin, & Nguyen, 2017). The fetus species of Campylobacter make sheep and cattle to have stillbirths. The fetus strain also leads to human infections (Nachamkin, & Nguyen, 2017).  The Campylobacter is a component of the micro molecules that make up the saliva. The bacterium causes itching of the gastrointestinal tract (Nachamkin, & Nguyen, 2017). This paper looks at the DNA based identification methods for Campylobacter. The paper explores the process of DNA extraction, PCR, and Gel Electrophoresis.

DNA Extraction

DNA extraction is the first procedure for identifying Campylobacter species. The first step is to take blood samples from individuals and poultry animals suspected to have the bacterium. A proper example is a child who has consistent diarrhea. Both chemical and physical methods are applicable in the extraction procedure. Usually, the first step of the extraction is to break plasma membrane to expose the possible DNA source to the diagnostic processes (Hoseinpour, Foroughi, Nomanpour, & Nasab, 2017). However, blood samples lack the thick membrane that should undergo crushing to retrieve the DNA source.  Therefore, the first procedure involves taking blood samples from infected person or animal.

Secondly, the blood sample is exposed to surfactants or even detergents. The two reagents dissolve and get rid of the nucleolus and the lipids in the sample. The detergents added should be directly proportional to the sample volume (Hoseinpour et al., 2017). The blood sample also contains protein and RNA. The researcher must get rid of the two kinds of molecules. DNA is a protein; consequently, the RNA is another protein. When RNA is not eliminated, the identification of the DNA becomes difficult since the procedure for identification detects all proteins in the sample (Eriksson, Mourkas, González-Acuna, Olsen, & Ellström, 2017).  Therefore, the addition of RNAses and proteases dissolves and removes RNAs and proteins respectively.

DNA Extraction for Campylobacter Identification

The resulting solution is exposed to a solution of salt that has high levels of anions and cations. The resolution of salt enables the remains of RNAs, lipids, and proteins to stick together. Afterward, the resulting solution is transferred to the centrifuge. The centrifugation separates DNA molecules from the mixture of the three particles. The resultant density of the mix differs from that of DNA. The combination of RNAs, lipids, and proteins has a higher frequency than the DNA molecules (Amadi et al., 2017). Therefore, DNA molecules float above the rest of the mixture. The two cannot form a uniform solution since the other components exist as a single solution.

The purification step follows centrifugation. The solutions mixing with the DNA such as the detergents must exit the answer to remain with pure DNA. The most common method of purification is by the use of isopropanol or any other suitable alkanol (Amadi et al., 2017). The isopropanol must be cold since high temperatures can denature the DNA. The proteinous nature of DNA makes it to unwound and loses its bases at elevated temperatures. DNA does not dissolve in alkanols hence remain as a precipitate as the other components of the solution dissolve in the isopropanol (Amadi et al., 2017). The second centrifugation procedure follows the purification process. The addition of sodium acetate improves the precipitation process.

The detection of the separated DNA follows the second centrifugation procedure. An indicator always detects whether the DNA molecules are in the sample or otherwise. The DNA solution undergoes the process of hydrolysis through a chemical means (Harzandi et al., 2015). Hydrolysis can be through heating of the sample in the presence of an acid.  The indicator such as diphenylamine reacts with DNA to form a bluish solution. In case the solution developed is not blue, then the Campylobacter is absent. However, individuals who have symptoms of the diseases that the bacterium causes must expect the blue coloration (Harzandi et al., 2015). After a successful extraction process, Polymerase Chain Reaction (PCR) is necessary to amplify the DNA.

Polymerase Chain Reactions (PCR)

The PCR method follows the extraction and identification of the Campylobacter DNA. The procedures amplify the DNA structures making them visible after electrophoresis process (Fukuda, Kojima, & Fujimoto, 2017). Amplification means the making of numerous copies from a double strand DNA structure. The process occurs inside a modern technology machine. The initial procedure involves the separation of the double strand DNA. Secondly, the researcher must allow the separate strands to cool down separately. The individual DNA templates then come into contact with the polymerases (De Boer, Rahaoui, Leer, Montijn, & Van der Vossen, 2015). The polymerases copy the structure of the strand to make complimentary copies. The primers are necessary for the amplification process to jump start.

Polymerase Chain Reaction (PCR) for Campylobacter Identification

The amplification process gears towards a given region in the DNA structure. Before the beginning of the process, multiple components and chemical must be present. The first component is the template. A specific region of the DNA should be in the model. The DNA polymerase is the second requirement. The enzyme catalyzes the amplification process under elevated temperatures. The process requires the presence of amplification. The primers should be two in number to complement both to the five prime and three prime ends (Van, Anwar, Scott, & Moore, 2018). Primers assist to jump-start the amplification process. The primers chosen must be specific to the target area in the DNA helix.

The fourth requirement is the deoxyribonucleotide. The compound should contain three phosphate groups. The dNTP acts as the foundation in which the polymerase forms another DNA structure from the original helix (Van et al., 2018). A solution that serves as a buffer is also a requirement. The solution creates a favorable reaction font for the activity of polymerases. Cations which are bivalent are necessary for DNA production. The cations include manganese and magnesium ions. Cations which are monovalent are also essential in the amplification process. Such ions include sodium, potassium and another viable group one ions (Laprade et al., 2016). The amplification process occurs at 100 microlitres when in smaller quantities (Laprade et al., 2016). However, 0.3 milliliters is ideal for reactions in the heat-sensitive cycler (Laprade et al., 2016).

The first process is called the initiation step. The step involves raising the temperature of the segment for the reaction through heating means. The method of increasing the temperature is necessary since the polymerases act best at elevated temperatures (Laprade et al., 2016). The initial heating should target the highs of 95 degrees Celsius. The second process involves denaturation. In step, heating continues for up to thirty minutes (Laprade et al., 2016). During the cooking, the temperature remains at 95 to 96 degrees Celsius (Laprade et al., 2016). The high temperatures force the DNA helix to melt. Moreover, the elevated temperatures break the DNA to form two separate complementary strands.

The annealing process follows the denaturation procedures. At this juncture, the researcher lowers the reaction temperature to 55 degrees Celsius (Laprade et al., 2016). The primers start to bond with the tail, and the head ends of the separated DNA molecules. The elongation process follows after the annealing of the primers to the ends of the DNA templates. The polymerase initiates the process. The enzyme requires high temperatures for optimal activities. Thus, temperatures of 76 degrees Celsius are necessary (Laprade et al., 2016). The polymerase creates another strand that complements the initial template. The polymerase then adds phosphate groups to enhance the elongation process. The resultant solution should then pass to get electrophoresis for viewing and further analysis.

Gel Electrophoresis for Campylobacter Identification

Gel Electrophoresis

After amplifying the DNA of the Campylobacter species, the next step is to run the sample in electrophoresis. The process enables an individual to gauge the quality of the DNA molecules. Additionally, a person can measure the intensity of their infections by checking at the results of electrophoresis. The method separates the DNA from other particles that remained during the extraction process (da Silva, Tejada, Blum-Menezes, Dias, & Timm, 2016). The process relies on the charge and the sizes of the molecules in the separation process. The electrophoresis field is charged to read negative to the cathode.

Electric currents run across the agarose gel hence charging it. Therefore, the first level of separation is regarding charges. The positively charged molecules move to the cathode, whereas, the negatively charged particles move towards the anode (Jordan, & Dalmasso, 2015). The movement of ions across the electric field is in respect to their weights. Therefore, more massive particles have slower rates of flow as compared to the smaller particles. The DNA molecules are smaller than the proteins, lipids and other impurities. Therefore, those impurities fail to reach the separation field leaving only the DNA molecules to undergo the process. The gel fractionates the DNA to purify the fragments further.

Gel electrophoresis then extends the DNA strands by cutting them out from the long helices. Ethidium bromide is necessary for the visualization of the DNA molecules to occur (Ozawa et al., 2016). The UV light illuminates the paths of the DNA particles hence enhancing the viewing process. Additionally, the Ethidium bromide stains the molecules to make them visible for viewing. The use of green dyes is necessary for improved visualization. The colorized agarose field containing the DNA undergoes illumination for clarity. During the visualization process, the composition of the DNA particles remains unchanged. Agarose process makes the recovery of DNA to be a straightforward procedure (Liu et al., 2017). Therefore, experts can take the DNA after the visualization procedure and use it for other diagnostic purposes.

The specialists should place the solutions that contain the DNA molecules in the material that comprises the gel. Additionally, an individual should transfer the gel into the chamber where electrophoresis should take place. Afterward, the researcher should switch on the power. The application of currents then follows. The observation is that smaller DNA particles migrate quicker than the bigger ones.  Electrophoresis is a force that moves the DNA particles via the matrix of the gel (Liu et al., 2017). The DNA molecules are first placed into the wells before the application of electricity. The particles then migrate on the agarose gel at different paces according to their sizes. The researcher determines the nature of Campylobacter infection according to the movement of the particles. Smaller molecules associate with minor infection while larger particles point to severe disease.

Conclusion

Campylobacter species majorly affects the poultry. However, humans get the infection when they feed on infected animals. The DNA based identification methods for the bacterium involves DNA extraction, PCR and finally, gel electrophoresis. The process of removal consists of the collecting of samples from infected humans or animals. The RNA and proteins which closely associate with the DNA molecules must be removed first. The proteases and the RNAes dissolve the two particles hence removing them. A solution of salt makes the impurities to stick together after which centrifugation commence to separate the DNA from the impurities. Precipitation follows to acquire real DNA sample. An indicator is useful in the detection of the DNA.

After extraction, the PCR method follows. The procedure involves initialization, denaturation, annealing and extension steps. Initialization consists in raising the temperature of the PCR chambers above 90 degree Celsius to activate polymerase enzyme. Denaturation involves the melting of DNA to form separate strands. During the annealing, experts lower the temperature to allow the primers to bind at the five and three primers end. The polymerase then starts copying the information on the stand. Extension involves the addition of phosphate groups into the DNA. After the PCR process, gel electrophoresis commences. The method enables the separation of DNA from other impurities. Moreover, the application of ethidium bromide and the UV rays illumination empowers an individual to identify the campylobacter’s DNA

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