Dry swab RT-PCR test

#GS3 #SCIENCE

CSIR-Cellular and Molecular Biology (CCMB) revolutionary dry swab direct Real-Time Polymerase Chain Reaction (RT-PCR) test, which allows for faster testing of COVID-19, is gaining fast acceptance.

  • Indian Council of Medical Research (ICMR) recently granted approval to CCMB to commercially use the game-changing technology of dry swab RNA-extraction-free coronavirus testing method. 
  • The method has potential to scale up testing by two to threefold with no additional resources and significantly reduce the time and costs of such tests.

 

Real-time PCR

  • Real-time polymerase chain reaction (real-time PCR) is commonly used to measure gene expression. 
  • It is more sensitive than microarrays in detecting small changes in expression but requires more input RNA and is less adaptable to high-throughput studies. 
  • It is best suited for studies of small subsets of genes. 
  • Its one major shortcoming is that the sequence of the specific target gene of interest must be known (so you can design the PCR primers), hence real-time PCR can only be used for studying known genes.

 

PCR Steps

  • The first step in a real-time PCR reaction is the conversion of RNA to complementary DNA (cDNA) – this process is known as reverse transcription. 
  • The next step uses fluorescent reporters and a PCR reaction to amplify and detect specific genes. 
  • Two types of fluorescent reporters are commonly used; these are SYBR green and Taqman probes.

 

SYBR Green & Taqman Probes

  • SYBR green is a dye that fluoresces only when bound to double stranded DNA (i.e the PCR product).
  • Taqman probes are made of a gene-specific nucleic acid probe, joined to reporter and quencher molecules.
  • The probe binds to the DNA between the forward and reverse primer. 
  • While the reporter and quencher are bound to the probe, the quencher absorbs the fluorescence emitted by the reporter. 
  • During the extension phase of the PCR reaction the probe is degraded, releasing the reporter and allowing its fluorescence to be detected. 
  • The advantage of the Taqman method is that probes with different coloured reporters can be combined in multiplex assays.
  • For both SYBR green and Taqman methods, the amount of fluorescence in a sample is detected in ‘real-time’ and plotted against the cycle number. 
  • The amount of fluorescence is proportional to the amount of PCR product. 
  • The time point at which the fluorescence reaches a defined threshold is relative to the level of gene expression. 
  • The design of real-time PCR experiments requires prior knowledge of the gene sequence and careful consideration of the types of controls to include.
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