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Coronavirus is frequent and rapidly spreading around the world, and in order to diagnose infectious diseases quickly, nucleic acid-based diagnosis has become an alternative to traditional culture- or immunoanalytic methods because of its speed and specificity.
in order to improve its sensitivity, current nucleic acid detection methods are usually targeted amplification prior to the detection step.
traditional amplification method is PCR-based and requires a thermal circulator for fine temperature regulation.
as an alternative to heat cycle-based amplification methods, isothermal amplification methods rely primarily on chain-replacement polymerases or T7 RNA polymerases at constant temperatures.
, however, due to the complex composition of iso-temperature amplification mixtures, these methods are often incompatible with detection methods, and the entire diagnostic process is usually a multi-step process.
-step diagnostics require additional time, instruments and reagents, and skilled personnel to perform the diagnostic procedures.
this aspect limits the wide applicability of nucleic acid diagnosis, especially where rapid and simple testing is required.
: Nasopharyngeal swabs were taken from patients with suspected COVID-19 and samples analyzed using standard rRT-PCR were obtained to confirm the presence of SARS-CoV-2.
the main mixture of isotherm reactions in a pot consists of the following components: 2 sl starting probe (10 sm), 2.2 sl reporting probe (10 sm), 5 sl peacock green solution (320 sm) (or 20 sl DFHBI-1T solution (50 sm), 10 sl NTP (25 mM each), 10 sl NTPs (25 mM each), 0.8 sl sl ET-SSB (500 ng m M), 0.8 μl ET-SSB (500 500 ng?l-1), 0.5?l recombinant ICT inhibitors (20 U l-1), 10 μl SplintR connective enzymes (25 U l-1), 5 slT7 RNA polymerases (50 U l-1) and 10 l 10×SENSR buffers (500 mM Tris HCl (pH 7.4) and 100 mM MgCl2).
the reaction master mixture to 99.22 sl in RNase-free water and add 0.78 sl target RNA to produce an overall product of 100 sl.
the reaction mixture at 37 degrees C for 0.5-2 hours.
culture, the fluorescence intensity is measured using the Hidex Sense 425-301 microplate reader (Hidex), as described above.
for peacock green fit, the background strength of the 100 sl 1×SENSR buffer containing 16 m peacock green is subtracted from all peacock stone green SENSR fluorescence intensity.
result: The assay can be performed in 30-50 minutes and can reach the limit of detecting RNA at 0.1 mole concentrations, relying on continuous iso-temperature reaction cascading reactions to produce RNA fittings that bind to fluorescent dyes.
T7 RNA polymerase transmeans RNA fittings from the connecting products of the promoter DNA probe and the reported molecular DNA probe, and the promoter DNA probe and the reported DNA probe interbreed with the target single-stranded RNA sequence through the SplintR connecting enzyme.
in 40 nasopharyngeal SARS-CoV-2 samples, the test achieved positive and negative predictions of 95% and 100%, respectively.
also shows that a range of viral and bacterial RNA can be detected quickly.
pan of thermostat RNA detection (SENSR) is a powerful diagnostic technique for RNA detection that provides short turnaround times, high sensitivity and specificity, and simple measurement steps without the need for expensive instruments and diagnostic specialists.
because of the simplicity and rapid development of the probe design process, SENSR will become the appropriate diagnostic method for emerging infectious diseases.
Woo, C.H., Jang, S., Shin, G. et al. Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription. Nat Biomed Eng (2020). MedSci Original Source: MedSci Original Copyright Notice: All text, images and audio and video materials on this website that indicate "Source: Mets Medicine" or "Source: MedSci Originals" are owned by Mets Medicine and are not authorized to be reproduced by any media, website or individual, and are authorized to be reproduced with the words "Source: Mets Medicine".
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