Table of contentsIntroductionDiagnostic testsDescription of diagnostic tests based on nucleic acidsIsothermal amplificationLAMPMicroarray-based technologiesNext generation sequencingConclusionIntroductionInfectious diseases are the main cause of mortality in recent times years. The World Health Organization (WHO) has said that around 13 million deaths worldwide are attributed to infectious diseases. The majority of these deaths are linked to people living in third world countries. These countries are more susceptible to the spread of these diseases because they are poverty-stricken. As funds are limited in these countries, basic health care is often of limited availability. Infectious diseases therefore spread more quickly in these countries. Everyone is at risk of contracting infectious diseases. Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get an original essay In recent years, more and more people are traveling, making it difficult to control these diseases. As more people move around the world, the complexity of these diseases has increased significantly. Along with the emergence of new pathogens, other diseases initially thought to be on the verge of extinction have re-emerged, such as multidrug-resistant tuberculosis. Other diseases classified as non-infectious have recently become infectious diseases. Helicobacter pylori is an example of an infectious disease. It has been noted worldwide as the leading cause of peptic ulcers and most likely gastric malignancies. Due to the increase in these diseases, diagnostic testing has become increasingly important. Diagnostic Tests An ideal diagnostic test has been proposed by WHO. This test is defined as “SAFE” (Affordable, Sensitive, Specific, User-friendly, Rapid, No Equipment and Delivered to those who need it). The perfect test would be able to run multiple tests at the same time with specific disease detection. With advances in technology, faster and more feasible tests have been implemented. These are known as nucleic acid-based diagnostics. These tests include the polymerase chain reaction (PCR) with reverse transcriptase 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 their importance in clinical and testing environments. Description of nucleic acid-based diagnostic tests. 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 powerhouse behind this mechanism is DNA polymerase. The “template” is the specific piece of DNA that is amplified. The primers are small and artificially create DNA sequences that bind to the template to create a site for the start of DNA replication. There are three essential steps that must be integrated. The first step is a denaturation step. The DNA helix is ​​subjected to intense heat in order to “unzip” the double helix into two single strands. The heater preventsenzymes to function in the mechanism. The next step is annealing. During this step, the temperature is lowered as the primers are able to find, detect and make bonds with the specific DNA polymerase, speeding up the creation of a DNA molecule making it match the arrangement of the matrix. The PCR technique is so specific because of the arrangement of primers that correspond to a specific region of the DNA template. The formation between the two primers is only amplified. The rest is not amplified. The DNA polymerase usually incorporated at the start of this test is not useful because it becomes useless at high temperatures. As that was a major inconvenience. The enzyme Taq polymerase was used instead. Isothermal amplification Isothermal amplification differs from PCR. For the PCR technique, various steps are required. Isothermal methods can be performed in a single step at a given temperature. Many techniques are designed around DNA replication. Some are designed on enzyme-based digestion or enzymatic nucleic acid assembly. Those that will be discussed are NASBA and LAMP. NASBA This technique is a method of sequence adaptation and is capable of maintaining itself. The reason for this diagnostic test is to amplify single strands of RNA by duplicating retroviral RNA. There are three enzymes essential to this test, “reverse transcriptase, RNase H and DNA-dependent RNA polymerase”. As the mechanism is executed, the targeted RNA is first transferred to the forward primer. From there, the RNA is modified into an identical transitional DNA by the use of the two enzymes “reverse transcriptase and RNase H”. Once this operation is completed, the double helix cDNA which consists of a promoter site is created by the second primer. Usually, 109 times amplification can be achieved in 1.5-2 hours at a temperature of around 41 degrees Celsius. LAMPAs discussed in the literature above NASBA is terminated at a temperature of 40 to 41 degrees Celsius or lower. This diagnostic test, unlike PCR, requires only one step. This testing technique is extremely specific and requires the use of four target-specific primers. This assay is incorporated for the analysis of amplified DNA sequences and for the detection of six specific regions of the DNA sequence that need to be analyzed in the sequences as shown in Figure 2. The required primers consist of a forward primer and reverse on the inside with two primers on the outside. Two steps are initiated when DNA polymerase starts the reaction with the mobility of the DNA strands. These two stages are defined as the starting structure production stage and the cyclic amplification stage. The DNA is removed by 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 each cycle. The final products of the reaction are stem-loop DNAs with the analyzed gene repeated inversely. The efficiency and specificity of this mechanism are increased due to the presence of a primer. There are by-products associated with the LAMP technique. These products are pyrophosphate ions. These ions form bonds with magnesium ions present in the reaction mixture to produce a white magnesium pyrophosphate precipitation clearly visible to the naked eye. Microarray-based technologies Although PCR and isothermal amplification are effective methods, they are slow and expensive to use although they are of great advantage. In order to overcome this drawback, other diagnostic tests have been developed. These are diagnostic tests based onmicrochips/chips. The purpose of this diagnostic test is to allow the analysis and analysis of numerous DNA sequences in a single test. This test is in complete contrast to the diagnostic tests mentioned above. It is for this reason that there is no requirement for an amplification step in this test. A test commonly known under this heading is Southern Blotting. This test involves placing DNA on a gel and separating it into small pieces of DNA sequences which are then placed securely on nitrocellulose filters. The separated small DNA fragments are then subjected to the hybridization process powered by radio or fluorescent probes. These probes have specific labels that are attached and can be detected by scanners specifically designed for this method. Next Generation SequencingAn advancement in the field of nucleic acidsA diagnostic test has been carried out with the involvement of Next Generation Sequencing (NGS). This changed the way scientists performed testing in the past. NSG testing has the ability to perform experiments that were not previously cost-effective or feasible. A variety of tests fall under this heading, such as the Roche (454) GS FLX sequencer and the Illumina genome analyzer. Roche (454) GS FLX sequencer The combination of emulsion PCR and pyrosequencing gave rise to this 454 technique. This method is a process using pyrosequencing first introduced in 2004. This process requires the release of a molecule pyrophosphate using the enzyme DNA polymerase. This causes a multitude of reactions leading to the production of light. This light production comes from the cleavage of oxyluciferin by luciferase. The amplification of the DNA strands is done by emulsion PCR. This is used to replace tubes and wells. There are thousands and thousands of oligomers on the top layer of these beads. This test works by using a combined mixture of oil and water to separate 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 mixture of separate strands of oligo-ligated adapter DNA fragments. This process is carried out by a microfluidic cluster station which allows the DNA fragments to be added to the top of the glass flow cell. Each of these cells is separated into 8 different pathways and the interior is covered with oligoes that correspond to the adapters. designed especially for them. The constant increase and decrease in heat and cooling causes DNA hybridization. DiscussionIn today's world, DNA analysis and amplification of genes by PCR has been made sufficiently easy due to the structural double helix design established by Watson and Crick. The PCR test is of valuable importance in the medical sector because it can detect hereditary diseases and be used for paternity testing. An advantage of the PCR technique is that it is a rapid and more refined detection method than previous antibody-based detection. The reason PCR is preferred over antibody-based detection testing is that humans susceptible to disease make antibodies to defend against infection. Unfortunately, it takes time for the body to produce these specific antibodies, which could lead to the patient not receiving the appropriate treatment. The consequences could lead to the spread of the infectious disease and, hypothetically, cause an epidemic. On the other hand, the PCR technique makes it possible to detect the disease in its early stages. The PCR technique has been used to detect diseases in human blood. The flu is.