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انواع تکنیک های مولکولی

Molecular methods can complement conventional culture-, antigen- and antibody-based methods for the detection, identification and epidemiologic analysis of infectious microorganisms. Disadvantages of conventional methods include their inability to distinguish new pathogens derived from a common ancestor, time-inefficiency while providing only satisfactory results and poor sensitivity and specificity in test results. Molecular methods are now emerging as important tools in all laboratories for routine detection and fingerprinting, as well as aiding in public health surveillance, which could potentially allow the rapid implementation of infection-control and intervention practice. The various forms of Polymerase chain reaction (PCR) are the most frequently used molecular diagnostic techniques in the diagnosis of infectious pathogens. In certain clinical situations, such as diagnosis of viral encephalitis or monitoring of cytomegalovirus (CMV) and Epstein–Barr virus (EBV) in organ transplant patients, PCR has transformed laboratory investigations because of its exquisite sensitivity and specificity, and is clearly the method of choice. PCR allows the amplification of millions of identical DNA copies from an originally small amount of pathogen genome in a clinical sample. In principal, the target DNA is first extracted then denatured at high temperature. Specific oligonucleotide primers are annealed to the DNA in a lower temperature, followed by an extension phase during which the DNA polymerase enzyme copies the template strand. This cycle is repeated, usually 30–40 times, resulting in millions of identical DNA copies. Ligase chain reaction (LCR) is a modification of PCR. In this method, two adjacent probes hybridize to one strand of the target DNA. The small gap between the two adjacent primers is recognized by a highly specific thermostable DNA ligase, and ligated to form a single probe. The ligated products then serve as templates for the amplification process. LCR allows the detection of only single base pair mutations, and it is very specific. The most common application of LCR is in the diagnostics of Chlamydia trachomatis in cervical and urine samples. In Nucleic acid sequence-based amplification (NASBA) analysis, which is performed in isothermal conditions, the target RNA is converted to double-stranded DNA by using T7 RNA polymerase, RNaseH, and a primer with a T7 promoter. The DNA acts as a template to produce multiple copies of RNA with a polarity opposite that of the target, which can be used for the production of additional DNA templates. NASBA is also suitable for DNA, with slight modifications in the early steps of the process. A recent example of NASBA technology in clinical diagnostics is a real-time multiplex NASBA assay for the detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens. The principle of Transcription-mediated amplification (TMA) is similar to NASBA, except that it uses the RNaseH activity of reverse transcriptase, whereas NASBA uses a separate enzyme for that. Due to the introduction of West Nile virus (WNV) in to the US since 1999, screening of primary WNV infection in blood donors was initiated. Currently, TMA is referred to as the most sensitive nucleic acid test commercially available for WNV infection, and is used on a large scale for blood donor screening in the US. Another clinical example of a large-scale and worldwide use of TMA technology is in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and urogenital swab specimens. Strand displacement amplification (SDA) is another isothermal amplification method. The first set of primers containing a restriction site is annealed to DNA template. Second primers are then annealed adjacent to the first ones and start amplification, after which restriction enzyme HincII is introduced in order to nick the synthesized DNA. An exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I starts the amplification again, displacing the newly synthesized strands. Similar to other isothermal amplification techniques, the amplification process of SDA is very efficient. SDA is used, for example, in the diagnosis of N. gonorrhoeae infection from female endocervical swabs and male first-void urine samples. The Fluorescence in situ hybridization (FISH) technique allows the detection of microbial nucleic acid directly from the sample (or cultured sample) without prior nucleic acid amplification. Briefly, the technique consists of specimen fixation on a microscope slide, hybridizing the prepared sample with a specific fluorescent-labelled probe, and visual detection of the hybridization with a fluorescent microscope. In medical microbiology, the technique is used, e.g. in determination of antibiotic resistance and detection of Helicobacter pylori from gastric biopsy specimens. Identifying bacteria from CSF, or Staphylococcus aureus from blood cultures. The line probe assay (LiPA) is another nucleic acid hybridization test, in which specific oligonucleotide probes are attached at known locations on a nitrocellulose strip as parallel lines and hybridized with biotin-labelled PCR products. One of the widely used LiPA applications is rapid detection of rifampicin resistance in Mycobacterium tuberculosis. The LiPA technique is also applied in the detection of antiviral drug-resistant mutations of HBV. aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108329

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