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Infectious diseases are the major cause of mortality in recent years. The World Health Organization (WHO) has stated that about 13 million deaths worldwide are attributed to infectious diseases. The majority of these deaths are related to the people living in third world countries. These countries are more susceptible to the spread of these diseases due to them being poverty stricken. As there are limited funds in these countries basic healthcare is often of limited availability. Therefore, infectious diseases are spread quicker in these countries. Everyone is at risk of contracting infectious diseases. In recent years more people are travelling which leads to the difficult challenge in controlling these diseases. As more and more people are relocating throughout the world the complexity of these diseases has increased immensely. Along with new pathogens emerging, other diseases that had been originally thought of being on the cusp of extinction have reemerged such as multidrug-resistant tuberculosis. Other diseases that have been classified as being non-infectious have recently become infectious diseases. Helicobacter pylori are an example of an infectious disease. It has been noted worldwide as the main cause behind peptic ulcers and quite possibly gastric malignancy. Due to the rise of these diseases diagnostic tests have become increasingly important. An ideal diagnostic test has been proposed by the WHO. This test is defined as “ASSURED” (Affordable, Sensitive, Specific, User friendly, Rapid, Equipment free and Delivered to those who need it). The perfect test would be able to run many tests at the one time with specific detection for diseases. With the advancement of technology quicker and more feasible tests have been established. These are known as nucleic acid based diagnostics. These tests include reverse-transcriptase Polymerase Chain Reaction (PCR) which is generally the most used. Other amplification methods such as nucleic acid sequencing based amplification (NASBA), Loop Mediated Isothermal Amplification(LAMP) and Microarray/chip based technologies. A new method has been introduced in recent years known as Next Generation Sequencing (NGS). In this essay the methods will be described, their advantages and disadvantages and there importance in clinical and testing environments.
Polymerase Chain Reaction PCR is the most commonly used diagnostic test in DNA detection. The main components are water, reaction buffer, forward primer, reverse primer, magnesium chloride, Taq polymerase and a template. The power house behind the mechanism is DNA polymerase. The “template” is the specific piece of DNA that is amplified. Primers are small and create DNA sequences artificially which makes a bond to the template to make a site for the start of DNA replication. There are three essential steps that have to be incorporated. The first step is a denaturation step. The DNA helix is put under intense heat in order to “unzip” the double helix into two single strands. The heating causes the enzymes to stop working in the mechanism. The next step is annealing. During this stage the temperature is lowered as the primers are able to find, detect and make bonds to the specific DNA polymerase speeds up the creation of DNA molecule matching it to the template arrangement. The PCR technique is so specific due to the arrangement of the primers that are corresponding to a specific region of the template DNA. The formation between both primers is amplified only. The rest are not amplified. The DNA polymerase usually incorporated in the beginning of this test is not useful as it becomes useless at high temperatures. As this was a major drawback. Taq polymerase enzyme was used instead.
Isothermal amplification differs from PCR. For the PCR technique there are a variety of steps required. Isothermal methods can be completed in one step at one temperature. Many techniques are designed around DNA replication. Some are designed on enzyme-based digestion or enzyme nucleic acid assembly. The ones that will be discussed are NASBA and LAMP. NASBA This technique is a method of sequence coping and is able to sustain itself. The reason for this diagnostic test is to amplify the RNA single strands by duplicating retroviral RNA. There are three enzymes that are essential for this test, “Reverse transcriptase, RNase H and DNA-dependent RNA polymerase”. As the mechanism is being run the RNA that is being targeted transfers first to the forward primer. From this the RNA is altered to an identical DNA transitional by the use of the two enzymes “reverse transcriptase and RNase H. Once this is complete the double helix cDNA which is made of a promoter site is created by the second primer. Usually a 109 fold amplification can be achieved in 1. 5-2hrs at a temperature of about 41 degrees Celsius.
As discussed in the above literature NASBA is completed at a temperature of 40-41 degree Celsius or below. This diagnostic test unlike PCR requires only but a single step. This testing technique is extremely specific and is required to use four target specific primers. This test is incorporated for the analysis of amplified DNA sequences is for the detection of six specific regions in the DNA sequence that need to be analyzed in the sequences as shown in figure 2. The primers that are required consist of a forward and reverse primer on the inside along with two primers on the outside. Two stages are initiated when DNA polymerase starts the reaction with the mobility of the DNA strands. These two steps are defined as starting structure producing step and cyclic amplification step. [image: ]Figure 3 LAMP mechanism The DNA is removed by the synthesis and by the function of the other primers the sequence is primed. This primed sequence is an excellent template for DNA synthesis. From this loop DNAs are formed and incorporated in every cycle. The end products of the reaction are stem looped DNAs with the gene that is being analyzed repeated inversely. The efficiency and specificity of this mechanism is increased due to their being a primer present. There are by products associated with the LAMP technique. These products are pyrophosphate ions. These ions form bonds with the magnesium ions present in the reaction mixture to produce a white magnesium pyrophosphate precipitation clearly visible to the naked eye.
Although both PCR and Isothermal amplification are effective methods they are slow and costly to use even though they are of great advantage. In order to overcome this drawback other diagnostic tests have been developed. These are Microarray/chip based diagnostic test. The purpose of this diagnostic test is that it allows the running and analyzing of many sequences of DNA in one test. This test is completely in contrast to the before mentioned diagnostic tests. This is in relation that there is no requirement for an amplification step in this test. A commonly known test under this heading is “Southern Blotting”. This test is involves the DNA being placed on a gel and separated out into smaller pieces of DNA sequences that are then securely placed on nitrocellulose filters. The small fragments of the DNA that were separated are then put through the process of hybridization powered by either radio- or fluorescent probes. These probes have specific labels that are attached and can be detected by the scanners that have been specifically designed for this method.
An advancement in nucleic acid based diagnostics test was made with the implication of next generation sequencing (NGS). This has changed how scientists have carried out tests in the past. The NSG tests have the ability to carry out experiments that were not cost effective or feasible to perform before. A variety of tests fall under this heading such as Roche (454) GS FLX sequencer and Illumina genome analyzer. Roche (454) GS FLX sequencer The combination of emulsion PCR and pyrosequencing brought forth this 454 technique. This method is a process using pyrosequencing first introduced in 2004. This process requires that a pyrophosphate molecule is releases by means of the enzyme DNA polymerase. This causes a multitude of reactions to take place finishing with the production of light. This production of light is derived from the cleavage of oxyluciferin by luciferase. Amplification of the DNA strands is done by emulsion PCR. This is used in replacement of tubes and wells. There are thousands upon thousands of oligomers on the top layer of these beads. The way in which this test works is by a combined mixture composing of oil and water in order to separate out certain beads that contain their own specific DNA fragments. Illumina Genome Analyzer The test is based on a “sequencing by synthesis” action. This technique requires a mix of separate strands of adaptor oligo-ligated DNA fragments. This process is carried out by a microfluidic cluster station which allows the DNA fragments being added to the top of the glass flow cell. Every one of these cells is separated out into 8 different lanes and the inside is coated by oligoes that match to the adaptors designed specifically for them. The constant increase and decrease in heat and cooling causes hybridization of the DNA. DiscussionIn todays’ world analysis and DNA amplification of genes by way of PCR has been made sufficiently easier thanks to the design of the structural double helix established by Watson and Crick.
The PCR test is of valuable importance in the medical sector as it can test for heredity diseases and be used for paternity test. An advantage of the PCR technique is that it is quick and more refined as a detection method than previous antibody-based detection. Why PCR is preferred over the antibody-based detection test is that humans who may contract a disease make antibodies to defend against infection. Unfortunately, the body takes time to make these specific antibodies in which time could lead to the patient not receiving correct treatment. Consequences of this could lead to the spread of the infectious disease and hypothetically cause an epidemic. In contract the PCR technique can detect the disease at its early stages. The PCR technique has been used to detect diseases in the human bloodstream. Influenza is a disease that is commonly talked about but no test has been produced for its detection. An example of an infectious disease in which PCR is useful in detecting is congenital virus infection in new born babies.
As previously mentioned PCR can be used to detect pathogens present in the blood. PCR can be used to detect diseases like Hepatitis B and C, HIV and Syphilis. Although this technique is very well equipped for detecting diseases certain new tests need to be introduced for detection of major diseases like Influenza.
NASBA diagnostic test can detect diseases with contain only a single stand of DNA. Such diseases include HIV type 1, foot and mouth disease and respiratory syndrome virus. Along with testing for infectious diseases, NASBA has certain layouts in place for detection of bacteria and fungi such as Salmonella enterica. NASBA is a relatively simple test to perform as it only requires the use of a water bath. This is extremely useful in countries where there are limited financial funds. Like NASBA, the LAMP diagnostic test is suitable for the detection of pathogens such as bacteria and viruses, and with the incorporation of a reverse transcriptase enzyme LAMP can amplify RNA. LAMP is extremely useful in so far as that it can be used to detect for food borne pathogens like Salmonella and Escherichia coli. LAMP has been used in clinical environments for the detection of SARS-CoV and Mycobacterium tuberculosis. Certain inhibitory elements present in clinical samples are less applicable to both the NASBA and LAMP tests as they would be to the PCR diagnostic test. Thus, this leads to a significant decrease in time and cost for the tests to be carried out. Although both of these tests are useful in their own way there are certain drawbacks to them. As there is an amplification step needed it still takes a bit of time to get the correct result required for the correct diagnosis and treatment to be administered. Microarray/chip based technologies are in complete contrast in relation to their use in a clinical environment. Both the PCR diagnostic tests and the isothermal amplification tests are very well equipped to be used in clinical environments. The most challenging diagnostic test is DNA microarray/chip based tests. Human microbiome is the main detection site in which this technique is attributed to.
Browns Laboratory were the first to produce a microarray test for the detection of gastrointestinal microbiota. As microarrays have a low detection limit it is not a suitable test to be carried out for detection of sensitivity. Sensitivity is a vital component that must be a priority when implementing a diagnostic test. An example of its poor sensitivity is detection for Influenza. The known detection limit for this disease is 10^3 copies/microliter. The sensitivity of a currently used microarray is about 50fM which is about 10^4 copies/microliter. This unfortunately doesn’t meet the requirement standard needed in clinics. There are still many kinks that need to be looked at before this test can be used full time for the detection of infectious diseases in humans. At the moment its sensitivity is too low for detection of said diseases and could possibly lead to wrongful diagnosis and treatment of the patient who has contracted a disease. The influences in which NSG tests have been applied to basic research indicate that they can be used for molecular diagnostic testing. However, certain issues need to be sorted before this can happen. The cost, robustness and accuracy will need to be refined. At the moment the cost for the Roche test is $600, 000 and $3500-4500 for Illumina Diagnostic test.
From the literature presented it is clear that there are certain precautions present that can help detect the presence of infectious diseases and can be quickly diagnosed and treated resulting in the prevention of a possible epidemic spread of the disease. Although these techniques are quite good at their assigned role the future of nucleic acid based diagnostic test for human syndromic infectious diseases clearly lies in the period of time in which it can be detected and the cost of running these test. With the development of new techniques and tests such as NSG we are closer to having a quick easy and workable test which will be available to everyone. It is clear that these tests are heading in that direction which in my opinion is extremely valuable.
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