Abstract
The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.
Highlights
The outbreak of novel Betacoronavirus, SARS-CoV-2, which began in Wuhan, China in December 2019, has spread rapidly to multiple countries as a global pandemic
All targeted gene primer/probe set, and Reverse transcription polymerase chain reaction (RT-PCR) reagents were tested in simplex reverse transcription polymerase chain reaction (rRT-PCR)
Thanks to the specific probes that were labelled with different fluorescence dyes (HEX, ROX, and Fluorescein amidites (FAM)), the gene amplifications can be identified in the same reaction tube by using different filters of the Applied BiosystemsTM, 7500 Fast Real-Time PCR System
Summary
The outbreak of novel Betacoronavirus, SARS-CoV-2, which began in Wuhan, China in December 2019, has spread rapidly to multiple countries as a global pandemic. As of March 10, 2021, about 135 million people were confirmed with SARS-CoV-2 infection, and three million were died (https://www.worldometers.info/coronavirus/#countries). The increasing number of infections worldwide necessitates the need for a less-invasive, reliable, and fast diagnostic tool [1]. To achieve this goal, several studies have tackled this challenge and these efforts have yielded several diagnostic kits. Many approaches have been proposed to detect SARS-CoV-2 virus in nasopharyngeal fluids such as multiplex RT-PCR [2, 3], CRISPR/Cas12 [4, 5], and CRISPR-Cas3 [6], lateral flow immunoassay [7], paper-based biomolecular sensors [8], SHERLOCK testing in one pot [9], DNA aptamer [10], loop-mediated isothermal
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