Conventional laboratory protocol of spin-column-based nucleic acid extraction for molecular diagnostics for COVID-19. Step 1: sample collection and transportation. Biological specimen types are collected by various collection procedures, such as a nasopharyngeal swab, nasal swab, and oropharyngeal swab, and transported to the centralized laboratories as soon as possible after collection. Of particular interest for molecular epidemiology analysis are those by which the samples can be collected most conveniently and effectively at the lowest cost. Step 2: heat deactivation. The viruses are deactivated by incubating the clinical sample at an elevated temperature to destabilize viral proteins and assemblies, rendering them incapable of infection during the downstream manual operation. Step 3: lysis/digestion. Lysis buffer contains a high concentration of chaotropic salts and detergents. Chaotropes disrupt hydrogen interactions and lead to the destabilization of proteins and nucleases. Organic amphipathic detergents break up the cell membrane structure by separating membrane proteins with the hydrophobic part of detergents from membranes. Proteases can be included in the lysis buffer to digest the contaminating proteins and degrade the nucleases. The lysis buffer shows higher efficiency at elevated temperatures. Step 4: binding. The lysate is transported to a spin column. The chaotropic salts provide favorable conditions for nucleic acid transfer to the silica membrane of the spin column by creating a hydrophobic environment to break down the association between NAs and water. Meanwhile, chaotropes provide positively charged cations to saturate the silica membrane, thus improving the absorption of negatively charged phosphate backbones of NAs under hydrophobic conditions. Since NAs are insoluble in ethanol, the addition of ethanol will enhance nucleic acid precipitation to the silica membrane. Step 5: wash the washing buffer, with ethanol as the dominant component, removes impurities such as protein polysaccharides residues. Multiple washing steps are usually conducted to thoroughly remove residual contaminations and buffer solutes. Residual ethanol should be avoided after the washing step since it may prevent the sequent elution of NAs and inhibit nucleic acid amplification. Step 6: elution. The elution buffer or pure water at pH 8-9 is typically used to release the NAs from the silica membrane to the bottom of the centrifuge tube during centrifugation. Step 7: reverse transcription-polymerase chain reaction (RT-PCR). The purified RNAs are reverse-transcribed to complementary DNA (cDNA), followed by cDNA amplification and fluorescence detection.
