Abstract
Background: COVID-19 infection is a major public health problem worldwide, and the D614G mutation enhances the infectivity of COVID-19. Methods: A probe-directed recombinase amplification (PDRA) assay was discussed to detect the D614G mutation at 39 ℃ for 30 min. The sensitivity, specificity, and reproducibility of the PDRA were evaluated by D614 and G614 recombinant plasmids. The clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative samples. Direct sequencing was carried out in parallel for comparison. Result: With good reproducibility and specificity, the PDRA assay worked well with the concentration in the range of 103–107 copies/reaction. Compared with direct sequencing as a reference, the recombinase-aided amplification (RAA) assay obtained 100% sensitivity and 100% specificity using clinical samples. Conclusions: A rapid, convenient, sensitive, and specific method to detect D614G mutation was developed, which offers a useful tool to monitor mutations in COVID-19 virus RNA.
Highlights
At the end of 2019, coronavirus disease 2019 (COVID-19) caused by COVID-19 virus, known as SARS-CoV-2, was first discovered and quickly began to spread around the world [1]
High-frequency mutations in the COVID-19 genome were found in nsp6, RNA polymerase, helicase, membrane glycoprotein, RNA primer, nucleocapsid phosphoprotein, and spike protein genes [2]
One of the most critical mutations was the D614G of the spike protein gene (S), which is a replacement of aspartic acid (D) with glycine (G)
Summary
At the end of 2019, coronavirus disease 2019 (COVID-19) caused by COVID-19 virus, known as SARS-CoV-2, was first discovered and quickly began to spread around the world [1]. Some researchers evaluated and compared the whole genome sequences of circulating COVID-19 and found mutations associated with the infectivity of the virus. High-frequency mutations in the COVID-19 genome were found in nsp, RNA polymerase, helicase, membrane glycoprotein, RNA primer, nucleocapsid phosphoprotein, and spike protein genes [2]. Many conventional methods are available for detecting single nucleotide polymorphism (SNP), such as real-time PCR [6], DNA sequencing [7], restriction fragment length polymorphism (RFLP) [8]. These methods are either time-consuming and laborious or require more sophisticated instruments and skillful personnel. COVID-19 infection is a major public health problem worldwide, and the D614G mutation enhances the infectivity of COVID-19
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