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polymerase chain reaction” is a highly accurate test used frequently in molecular medicines to diagnose any type of genetic material abnormalities. It is helpful in detecting any types of genetic abnormalities as well as infectious diseases. This technique can make several copies of DNA from any region even if the quantity of target DNA is negligible. PCR is well known as the “DNA amplification technique” as it can make several copies of target DNAs using the sequence (Green and Sambrook, 2019). Taq polymerase is isolated from heat-stable bacteria named “Thermusaquaticus”, which is the most essential component of this PCR. Taq polymerase enzyme uses the single-strand DNA as the template in order to make new DNA strands. A DNA primer is also very essential because this is a very short sequence of some nucleotides that acts as the initiator point of DNA synthesis (Sreejithet al. 2018). Three consecutive steps make millions and \billions of DNA copies. Following completion of this whole procedure, it can be visualised by the “Gel Electrophoresis” process. The most common gel electrophoresis which is used in this PCR is “agarose gel electrophoresis” (Koshyet al. 2017). Based on the size, charge as well as a confirmation the DNA can be separated in the gel. It is very useful for any sensitive detection of disease-causing microorganisms and DNA analysis of crime scenes or for investigating any archaeological specimens.
The aim of this assignment is to test the “Genomic DNA” from Omni Swabs and then amplify them using the PCR for further investigation.
In order to extract the target DNA from the Omni Swabs of biopsy material “the spin column method” has been used. Following the separation of the targeted DNA, the PCR has been performed to amply various copies of this targeted DNA. A master Mix has been used that contains the essential reagents for PCR such as MgCl2, dNTP, and Taq polymerase, as well as a buffer. This master mix allows the researchers to set up all the controls as well as test samples of the targeted genetics material, here the DNA without the need of measuring essential materials of PCR every time with accurate measurements. In the first step of the PCR, denaturation of targeted DNA has been performed at 94-96°C. (Cao et al. 2017) The very next step of getting the single-stranded DNA template from denaturation is annealing which has been done at approximately 55-66°C and is done using primers. In this PCR, mainly four types of forward and reverse primers have been used and these are “CYP2D6 A”, “CYP2D6 B”, “CYPIAI IV (ile-val)”, and “CYPIAI''. The Taq polymerase that has been used in the master mix helped in the extension of the DNA strand by adding new nucleotides and it has been done at approximately 72°C. The result was analysed using agarose gel electrophoresis. There are four types of restriction digestions that have been used as well in the master mix that helped in producing compatible ends on the entire amplified DNA.
The amplified DNA can be visualised in the agarose gel and DNA fragments that have the same strength can form a “band” which can be identified using a type of DNA-binding dye such as “Ethidium bromide”. Using the DNA ladder the standards can be loaded in the gel and in a single and several copies of DNA are present with the same mass or numbers of base pair. The result of the tests using four different primers and four different restriction digestions is in the following section.
Agarose gel electrophoresis is the most efficient test for the detection of genetic material such as DNA. It has been found that if a higher-strength gel is used with larger concentrations, it is quite useful in the detection of larger DNA fragments (Allen et al. 2019). Moreover, it has been found that the more the concentration of the agarose gel is, the smaller the pore sizes will become. It has been found in the results that DNAs which are smaller than or equal to 20BP cannot be detected. According to the study by Zhang et al. (2020), the DNAs which are smaller than 20Bp can only be detected if using the Pulse field where the alternative current has to be used in two different directions (Yilmaz et al. 2017). It can be stated that the smaller the size of the DNA fragments, the more easily they travel through the pores of the gel and form the DNA fragments which can be visible in the form of bands (Bhattacharya and Van Meir, 2019). Moreover, if the pulse is used that helps in faster travel through the gel. As per the result, there are many DNAs which are larger in size. In the first result, the DNA ladder which has been used for reference has helped to detect the DNA lengths of the sample. In the first result, the standard or reference DNA fragments used ranged from 50 BP to 587BP. Hence, it can be stated that the minimum base pair or BP of the DNA that can be detected in this electrophoresis is 50 and the maximum length is 587. This helped to detect the band of 80BP, 168BP, and 188Bp. Therefore, it can be stated that in each band or fragments there are several DNAs that have 80, 168 and 188 base pairs in their structures. In this way, the targeted DNA can easily be identified and they can even be isolated from the gel.
It can be concluded that PCR is a very useful technique in the detection of mutant DNA. The mutant DNA could be identified in this experiment. Based on the various Base pair lengths of the DNAs, the smaller ones have travelled more and have created bands and the larger ones are present in the upper regions of the gel. Various primes have been used and based on that master mixes were prepared that contained restricted digestive particles. These helped in fragmenting larger-sized DNAs and were detected in the gel. PCR is certainly a useful technique in the detection of cancer or any other genetic disorder due to genetic mutation.
“Enzyme-linked immunosorbent assay” or ELISA is one of the very reliable methods for detection of sensitive proteins such as antigens, antibodies as well as some hormones and glycogens. There are mainly three types of ELISA and the underlying principle for ELISA is the specific antibody used in this test binds to the target antigens or proteins (Brangelet al. 2018). This is a quantitative assay where the amount of antibody binding provides a reliable measurement for the concentration of antigen-antibody concentration. This test is frequently used in the detection of serum antibodies as part of a viral test (Di Domenicoet al. 2021). On the other hand, this test can be used in food industries in order to analyse the presence of any types of food allergens. This is a rapid test and comparatively a less complicated process (Kim et al. 2021). Furthermore, for this test, an anti-human IgA antibody has been used in the microwell and the standards, control as well as unknown samples have been added for the quantitative analysis or for the measurement of the concentration of the unknown sample.
In the case of direct ELISA and indirect ELISA, the antigens are coated in the ELISA plates and this is then incubated for approximately one hour at room temperature or 37 degrees C. An alternative way to incubate this is at 4°C. However, for this test, the direct ELISA has been used and here at first the plates or microwells have been coated with the anti-human IgA antibodies for this test (Hosseini et al. 2018). On the other hand, the samples, control as well as standards have been added. Hence, if any human antibody is present in the sample, it will bind to the anti-human antibodies. In the first step of this test, the Anti-human IgA antibody is adsorbed on the microwell surface and following the addition of samples, standards or control. After the microwells are washed properly the enzyme “HRP-conjugate” has been added (Clark and Engvall, 2018). “HRP-conjugated anti-IgA antibody” will bind to if there is a presence of “IgA-anti-IgA” complexes. This ultimately results in the change in the colour of the microwells.
In order to prepare the standards for this test “Two-fold” dilution has been used. In S1 or standard 1, the concentration of the target protein or human IgA is 100 ng/ml. Therefore, the concentration of the S2 is 50 ng/ml. S3 is 25 ng/ml and this procedure has been repeated until the concentration reached 1.56 for S7. The concentration of protein in the blank test tube is 0 ng/ml.
In order to measure the protein concentration in the unknown samples A and B, the standard protein curve has been made in Excel. The concentration of the protein can be measured if measuring the absorbancy in the colourimeter or spectrophotometer. The OD or Optical Density decided for this test is 450nm. The absorbancy for the blank is 0.06 AU. The S1, S2, S3, S4, S5, S6, and S7 with the protein concentration are 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.125 ng/ml, 1.56 ng/ml. Based on the protein concentrations, the AU has been measured which are 0.067, 0.062, 0.067, 0.06, 0.056, 0.58, and 0.049. The absorbencies of Sample A (1, 2, 3) and Sample B (1, 2, 3) are 0.08, 0.134, 0.102, 0.081, 0.067 and 0.045. Based on the Standard curve the protein concentration of the unknown samples has been detected. The formula that has been used in the detection of protein concentration of unknown samples is [(AU-Slope)/Intercept]. The protein concentration of Sample A (1, 2, 3) and Sample B (1, 2, 3) are 1.403508772, 2.350877193, 1.789473684, 1.421052632, 1.175438596, and 0.789473684 respectively.
From the above graph, it can be seen that the concentration of sample A is 1.847953216 ng/ml. The concentration of unknown sample A has been plotted and as per figure 6, the concentration lies close to the protein concentration of S7. The graph has been plotted based on protein concentration and absorbency values and the protein concentration of Standard samples, and the concentration of the unknown sample lies at 1.85ng/ml protein concentration.
From the above graph, it can be seen that concentration of sample B is 1.128654971 ng/ml. The concentration of unknown sample B has been plotted and as per figure 7, the concentration lies close to the protein concentration of S7. The graph has been plotted based on protein concentration and absorbency values and the protein concentration of Standard samples, and the concentration of unknown samples lies at 1.13ng/ml protein concentration.
The standard is useful in representing the relationship between two variables and in those cases the protein or IgA concentration and their absorbancy have been considered. The more the concentration of the protein, the more is the absorbancy value and based on the plotted values, the trend-line can be achieved (Korasick and Tanner, 2018). Therefore, it can be stated that the standard curve is a very useful technique for the detection of protein concentration of any unknown sample from absorbancy. In order to detect the concentration of the IgA the direct ELISA technique has been considered in this test (Manalac et al. 2020). The change in the colour of the direct ELISA following the addition of HRP conjugate is either can be due to oxidation of the substrates or due to hydrolysis of phosphate groups of the substrates in presence of the substrate such as AP or Alkaline Phosphatase (AP). This direct ELISA that includes the rapid screening of the antibodies is very useful in identifying the HIV or “Human immunodeficiency virus”, any other types of pathogenic microorganisms like bacteria, fungi, viruses and even food allergens. It is a scientific technique that is used for the systematic detection of the above-mentioned cases (Cook et al. 2021). Due to that reason, it can be stated this test is very significant for the test of “human IgA antibodies”.
From the above evaluation, it can be stated that mainly the ELISA test has been tested here for testing “human IgA antibodies”. ELISA is considered a very reliable experiment for the identification of sensitive proteins like antigens, antibodies as well as different types of hormones and glycogens. This is mainly a rapid test and definitely a less difficult process. Hence, from the above evaluation, it can be inferred that the average protein concentration in the unknown sample has resulted in 1.84 and 1.13 and ng/ml which is very close to the protein concentration of standard 7. However, it can be also stated that the concentration of the protein is mainly measured for measuring the significant absorbancy in the colourimeter or in the spectrophotometer. Along with this, the microwells are mainly washed adequately with the enzyme “HRP-conjugate” which has been counted. It can be also concluded that “HRP-conjugated anti-IgA antibody” significantly binds to the existence of “IgA-anti-IgA” complexes.
Western blotting is considered to be a very useful technique that is used in the separation of various protein samples. It is very useful in the detection of target or specific proteins from a complex mixture of proteins based on their molecular size and weight (Kowal et al. 2017). Proteins of various types that are separated through the electrophoresis can be transferred into the blotting membrane that contains bases of various proteins. Following this step, the antibodies are added that can bind the protein of interest. The unbound antibodies or those that could not bind the targeted protein get washed off (Kumar et al. 2018). This is the reason that In this way those antibodies that could bind to specific proteins can only be visible on the film and based on the thickness of the bands, the concentration of proteins can be easily identified. Hence, if the standards can be used the western blotting technique can easily.
Disease due to Staphylococcus aureus is mainly a foodborne illness. In this case, intoxication due to the staphylococcus aureus is very common but the outbreak of this disease condition is mainly for a very short period of time and a very sudden type of occurrence. Therefore the outbreak in the maximum time cannot be detected due to the very short time. MRSA is the methicillin-resistant Staphylococcus aureus that is the S. aureus organism that is resistant to the methicillin group of the antimicrobial agent. That means the treatment procedure of the Staphylococcal foodborne disease generally cannot include the group of methicillin drugs as the treatment, in this case, is resistant to this drug of choice. There is another group of staphylococcal organisms that are susceptible to the group of methicillin drugs which means the groups of organisms respond to the medicines that are used to treat the diseases. These are called MSSA or “methicillin-susceptible Staphylococcus aureus” type of bacteria that responds well to medications used to treat staphylococcus infections.
The sample was collected with a swab from the nasal cavity of two sample groups (Krismer, 2017). This sample was first spread on a selective agar media (Mannitol-salt Agar media). Then the isolated organisms are purified on the BHI (brain Heart Fusion media). The BHI is a very nutrient-reach media for the purified growth of microorganisms.
In this case, to perform the gram staining a slide is taken and a loop full of sterile water or sterile saline (PBS) and a colony from the growth media are placed on the slide and emulsified. This process is performed for both samples differently. The emulsified mixing is spread out to make a thin film on the slide. The slide is Stained following Gram’s stain method using crystal violet as the gram stain and viewed under the microscope (Public Health England, 2014).
This is mainly performed to extract the DNA and carry on a genomic DNA experiment of the culture of S. aureus. In this case, centrifugation of the suspension of the sample at 20,000 X g for 1 min, and thenthe supernatant will be used as a template in each PCR reaction to amplify the DNA (McClure, 2006).
Analyze PCR products by gel electrophoresis
Agarose-gel-electrophoresis is performed to analyse the PCR products. By the use of dyes such as ethidium-bromide or non-toxic dyes, the result is visualized.
Spa typing PCR
This procedure is mainly performed to differentiate between the different types of strain (SPA, spaserver.ridom).
Multiplex 16S / TSST/ mecA PCR
This test is mainly an indicative test for the mecA positive of S. aureus. This test indicates a higher rate of MRSA incidences.
Agglutination assay to confirm S. aureusand identify MRSA
The agglutination assays are mainly performed to identify and differentiate the strain of the MRSA. A positive reaction is indicated by a firm clot that does not move generally when the tube was tipped (Omoshabaet al. 2020) .
In this case, the result shows the appearance of the yellow and round-shaped colonies in both samples of the patient and the staff. Gram stain shows the appearance of the purple cluster in the patient sample which says it is a grams positive organism and in the staff’s sample, the gram-staining shows a pink-colored organism which means the presence of the gram-negative organism. Agglutination assay shows negative results indicating the load of bacteria is lower. PBP2 agglutination assay shows a positive result for both staff and patient samples, indicating there may be coagulase-negative MRSA present. Spa-PCR shows a result of positive and 445bp for the patient’s case. The presence of 16S RNA is negative for both while the test-product and me-product results showed positive for the patient only.
In this experiment to detect the outbreak chances in the area, several tests were done based on the main two types of samples which include the patient of the health care organization group and the staff of the organization (HallinMet al. 2009). Samples were collected from the nasal cavity accordingly from them to perform different types of experiments to identify the presence of the bacteria. As the primary and mandatory steps the colonies were grown in the selective media from the collected sample of different two groups and then the colonies were purified in the BHI media (Krismer, 2017). And then gram staining is performed to detect the presence of the organism.
Figure 10 explains a standard procedure for streak plates in order to isolate Staphylococcus colonies. The template preparation was performed to carry out the process of DNA extraction. Therefore it is performed in the amplification of the DNA from each type of sample (McClure, 2006). In the Analyse PCR products by gel electrophoresis, after running the gel, the band intensity is used to estimate the quality and the quantity of the product. It gives an idea of a given molecular weight that is relative to the ladder of the electrophoresis. It has been seen that the maximum of the test of Multiplex 16S / TSST/ mecAPCRshows the presence of MRSA strains in a mecA test of S aureus. The MRSA contains a type of A or Spa-gene protein that is the virulent factor in it, which makes it different from other strains (SPA, spaserver.ridom). This procedure helps in discriminating among the different strains. The coagulation test, in this case, can be detected by both tube coagulase and slide coagulase and a positive result indicate the presence of S. aureus.
Figure 11 explains the staining of gram-positive bacteria. Here the colonisation of Staphylococcus aureus has been shown. Therefore, it can be stated that as per the result the group of patients shows a gram-positive outcome that indicates the presence of the gram-positive organism, MRSA and MSSA both are gram-positive strains of bacteria. The “Basic Local Alignment Search Tool (BLAST)” finds areas of local similarity between sequences.
This program corresponds nucleotide or protein sequences to sequence databases as well as calculates the statistical implication of matches. In the group of patients, the positive result shows the maximum chance of the presence of S aureus. Results of mastaStaph agglutination and PBP2 agglutination indicate the presence of non-coagulate staphylococcus organisms. This is finalized by the result of the Spa-PCR test which indicated a positive for the presence of MRSA and a load of 445bp (SPA, spaserver.ridom). mecAproduct-test finally confirms the presence of methicillin resistance S. aureus in the group of patients’ samples (Omoshabaet al. 2020).
According to the report and study by the World Health organization, the strain of MRSA is more virulent than MSSA. Therefore, the health organizations and the scientists are conscious of the outbreak of Staphylococcus aureus as this group of organisms is resistant to most of the available treatment procedures and causes severe bacteremia due to the secretion of enterotoxin A. This report helps to identify and detect the possibility of the outbreak of the S. aureus-associated food-borne illnesses. The outbreak due to s. aureus may become very severe because the outbreak is a short period of time. Due to the resistance of the maximum of the treatment procedure, it is very important to identify and differentiate the strain from the other strains of the S.aureus. From this report, it can be concluded that the sample collected from the group of patients shows a positive result for the presence of S. aureus which indicates a possible chance of an outbreak of the S. aureus-associated disease.
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