IndexIntroductionDiagnostic testsDescription of diagnostic tests based on nucleic acidsIsothermal amplificationLAMPMicroarray/chip based technologiesNew generation sequencingConclusionIntroductionInfectious diseases are the main cause of mortality in recent years. The World Health Organization (WHO) said that approximately 13 million deaths worldwide are attributed to infectious diseases. Most 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 affected by poverty. Because funds are limited in these countries, primary healthcare is often in limited availability. Therefore, infectious diseases spread more rapidly 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 Shouldn't Be Banned"? Get an original essay In recent years, more and more people are traveling, which leads to the difficult challenge of controlling these diseases. As more and more people move around the world, the complexity of these diseases has increased immensely. Along with the emergence of new pathogens, other diseases that were originally 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 cancer. Due to the increase in these diseases, diagnostic tests have become increasingly important. Diagnostic Tests WHO has proposed an ideal diagnostic test. This test is defined as “ASSURED” (convenient, sensitive, specific, easy to use, rapid, without equipment and delivered to those who need it). The perfect test would be able to run multiple tests at once with specific disease detection. With the advancement of technology, faster 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 widely used. Other amplification methods such as nucleic acid sequencing-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and microarray/chip-based technologies. In recent years, a new method known as Next Generation Sequencing (NGS) has been introduced. In this essay we will describe the methods, 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 the mechanism is DNA polymerase. The “template” is the specific piece of DNA that is amplified. Primers are small and artificially create DNA sequences that bind to the template to create a site for DNA replication to begin. There are three essential steps that need to be incorporated. The first step is a denaturation phase. The DNA helix is ​​subjected to intense heat to "unzip" the double helix into two single strands. Heating causes the enzymes to stop working in the mechanism. The next step is annealing. During this step the temperature is lowered as the primers are able to find, detect and bond with the specificationDNA polymerase, accelerating the creation of the DNA molecule by adapting it to the template arrangement. The PCR technique is so specific due to the arrangement of primers that correspond to a specific region of the template DNA. The formation between the two primers is only amplified. The rest is not amplified. The DNA polymerase usually incorporated at the beginning of this test is not useful as it becomes useless at high temperatures. Since this was a major inconvenience. The enzyme Taq polymerase was used instead. Isothermal Amplification Isothermal amplification differs from PCR. Several steps are required for the PCR technique. Isothermal methods can be completed in a single step at one temperature. Many techniques are designed around DNA replication. Some are designed around enzyme-based digestion or enzymatic nucleic acid assembly. The ones we will talk about are NASBA and LAMP. NASBA This technique is a sequencing method and is capable of supporting itself. The purpose of this diagnostic test is to amplify single strands of RNA by duplicating the retroviral RNA. There are three enzymes essential for this test: “Reverse transcriptase, RNase H and DNA-dependent RNA polymerase”. As the mechanism runs, the targeted RNA first transfers to the forward primer. From this the RNA is modified into an identical transitional DNA by the use of the two enzymes “reverse transcriptase and RNase H. Once this is completed, the double-stranded cDNA consisting of a promoter site is created by the second primer. It is usually possible to achieve 109-fold amplification in 1.5-2 hours at a temperature of about 41 degrees Celsius. The LAMPAs discussed in the above NASBA literature are completed at a temperature of 40-41 degrees Celsius or lower. This diagnostic test, unlike PCR, requires only a single step. This testing technique is extremely specific and requires the use of four target-specific primers. This assay is incorporated for the analysis of the amplified DNA sequences and for the detection of six specific regions in the DNA sequence that need to be analyzed in the sequences shown in Figure 2. The required primers consist of a forward and a reverse primer on l 'inside along with two primers on the outside. Two phases are initiated when DNA polymerase initiates the reaction with the mobility of the DNA strands. These two steps are defined as the initial structure production phase and the cyclic amplification phase. The DNA is removed from the synthesis and based on the function of the other primers the sequence is triggered. This primed sequence is an excellent template for DNA synthesis. From this loop, DNA is formed and incorporated into each cycle. The final products of the reaction are stem-loop DNA with the gene being analyzed repeated in reverse. The efficiency and specificity of this mechanism are increased thanks to the presence of a primer. There are 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 precipitation of white magnesium pyrophosphate clearly visible to the naked eye. Microarray/chip-based technologies Although both PCR and isothermal amplification are effective methods, they are slow and expensive to use even though they are of great benefit. To overcome this drawback, other diagnostic tests have been developed. These are microarray/chip-based diagnostic tests. The purpose of this diagnostic test is to allow the performance and analysis of many DNA sequences in a single test. This test is completely in contrast to the diagnostic tests mentioned above. This is due to the fact that an amplification step is not required in this assay. A testcommonly known under this title is “Southern Blotting”. This test involves DNA being placed on a gel and separated into smaller pieces of DNA sequences which are then securely placed onto nitrocellulose filters. The small fragments of DNA that have been separated are then subjected to the hybridization process powered by radio or fluorescent probes. These probes have specific labels attached and can be detected by scanners specially designed for this method. Next Generation Sequencing An advance in the field of nucleic acids has been a diagnostic test with the implication of next generation sequencing (NGS). This has changed the way scientists have done testing in the past. NSG testing has the ability to perform experiments that were previously not convenient or feasible to perform. A variety of tests fall into this category, such as Roche's GS FLX sequencer (454) and the Illumina genome analyzer. Roche GS FLX Sequencer (454) The combination of emulsion PCR and pyrosequencing brought this 454 technique to light. This method is a process using pyrosequencing first introduced in 2004. This process requires a pyrophosphate molecule to be released by the enzyme DNA polymerase. This causes a multitude of reactions that end in the production of light. This light production results from the cleavage of oxyluciferin by luciferase. Amplification of DNA strands is carried out by emulsion PCR. It is used to replace pipes and manholes. There are thousands and thousands of oligomers on the top layer of these spheres. The way this test works is by using a combined mixture of oil and water to separate certain spheres 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 separated strands of oligo-ligated adapter DNA fragments. This process is performed by a microfluidic cluster station that allows the addition of DNA fragments to the top of the glass flow cell. Each of these cells is separated into 8 different lanes and the inside is lined with oligos that correspond to the adapters designed specifically for them. The constant increase and decrease of heat and cooling causes DNA hybridization. Discussion In today's world, DNA analysis and amplification of genes by PCR have been made sufficiently simpler due to the design of the structural double helix established by Watson and Crick. The PCR test is of valuable importance in the medical industry as it can test for 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 who might contract a disease produce antibodies to defend themselves against infection. Unfortunately, it takes time for the body to produce these specific antibodies, and time may lead to the patient not receiving the correct treatment. The 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 in its early stages. The PCR technique has been used to detect diseases in the human bloodstream. Influenza is a commonly talked about disease, but no test has been produced to detect it. An example of an infectious disease in which PCR is useful to detect is congenital viral infection in newborns. As mentioned above, PCR can be used for.