Parasitie analysis via Molecular Biochemical Technique PCR: The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. The Method: Despite the slow start on developing these assays, the further progress and utilization of nucleic acid-based assays to detect parasitic pathogens can and will play a role in the epidemiology, prevention, and treatment of parasitic diseases. In this review, only assays that have been described in the literature during the last 5 years will be discussed. Nucleic acid-based probe detection of parasitic agents consists of the use of a reporter DNA molecule to detect specific parasite DNA or RNA sequences. The parasite within the target specimen is lysed with a membrane perturbant such as alkali, detergent, heat, chaotropic agents (urea and guanidine hydrochloride), or sonic disruption, and the nucleic acid is liberated and then denatured. The target sequence is detected by the reporter molecule following successful hybridization. The reporter molecule can be composed of an oligonucleotide, a DNA fragment, single-stranded DNA, or plasmid DNA. A label consisting of a radionucleotide, enzyme, antigenic molecule, affinity label, or chemiluminescent substrate is attached to the reporter. A positive signal is generated directly or indirectly by the reporter molecules that hybridized to target sequences in solution or immobilized on solid supports, such as filter paper, nitrocellulose, and nylon membranes. In 1985, a strategy for amplifying target DNA sequences via in vitro DNA replication was described (102). PCR utilizes two oligonucleotides which flank the target sequence and a DNA polymerase, in successive cycles of denaturation and hybridization with oligonucleotide primers, followed by extension of the primers by action of the polymerase to generate billions of copies of the target sequence. The amplified target sequence is detected by probes as described above, or the DNA product can be analyzed directly by gel electrophoresis. Currently, the detection and diagnosis of parasite infections rely on several methods in addition to clinical symptoms, clinical history, travel history, and geographic location of patient. Each diagnostic method has inherent advantages and disadvantages, irrespective of the type of parasite or clinical specimen being tested. The chief advantages of nucleic acid-based detection techniques are their sensitivity for detecting pathogens and the speed at which they can definitely identify an organism. If culture or animal inoculation is required for the identification of the parasite and the introduction of therapy, then probe detection or PCR offers an advantage. When direct microscopy is sufficient for parasite detection and species identification by morphology, and the level of parasite is sufficiently high, then nucleic acid-based technology is not advantageous except for processing a large number of specimens with an automated assay. However, when the parasite load is low, then a sensitive diagnostic test involving nucleic acids is beneficial. Serologic detection of antibodies to parasites is useful as a screening device, but often there are cross-reactive antigens compromising specificity, and serology generally does not discriminate between current active infection and either prior or latent infection. The results with nucleic acid-based assays are independent of immunocompetence or previous clinical history and can distinguish between organisms that are morphologically similar and/or share antigenic epitopes, and the organisms do not need to be viable or culturable. An inherent disadvantage of these assays is that isolates containing variant DNA sequences may be missed even though the sequence is not associated with virulence.
Posted on: Thu, 28 Aug 2014 12:34:51 +0000
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