4.5. Sewage sampling and pretreatment via the Shennong-1 robot
The Shennong-1 robot consists of several functional modules, including automatic sewage sampling, automatic enrichment and concentration, automatic nucleic acid extraction, and automatic reverse transcription. In Shenzhen Third People’s Hospital, the robots were installed in tributaries receiving sewage, mainly from areas where patients infected with Omicron BA.5 were isolated for treatment.
Enrichment and concentration of Omicron BA.5 in sewage was carried out following the aluminum hydroxide adsorption−precipitation method recommended by the standard WS/T 799-2022. Briefly, a 50 mL sewage sample was centrifuged at 2500 g for 30 min at 4°C. Then, 39.5 mL of supernatant, 0.5 mL of aluminum chloride solution (0.3 M), and 10 mL of PBS buffer (0.1 M, pH 6.4) were added to a 50 mL centrifuge tube and mixed evenly to form an aluminum hydroxide colloid. After 15 min of incubation at room temperature, centrifugation was performed at 1900 g for 5 min at 4°C. The supernatant was discarded, and 0.20 g ± 0.01 g of disodium EDTA dihydrate was added to the remaining colloid. After treatment at 60°C for 6 min in a water bath, the resulting liquid with a volume of approximately 1 mL was obtained for RNA extraction.
The viral RNA extraction was performed using a nucleic acid extraction kit in accordance with the instructions provided by the manufacturer. Briefly, 15 µL of protease K, 500 µL of lysis solution, and 4 µL of magnetic bead solution were added to 200 µL of concentrated sewage, and the mixture was incubated at 65°C for 4 min. Subsequently, RNA was magnetically adsorbed for 1 min, after which the magnetic beads were washed with 600 µL detergent for 30 seconds. Finally, after RNA was washed off the magnetic beads by 100 µL eluent at 80°C over the course of 2 min, the magnetic beads were removed to obtain an RNA-containing solution.
The reverse transcription of RNA extracted was conducted using the PrimeScript™ RT reagent kit (Takara, Dalian, China), following the instructions provided by the manufacturer. To begin, a mixture comprising 10 μL of 5× gDNA (genomic DNA) Eraser Buffer, 5 μL of gDNA Eraser, and 35 μL of RNA template was prepared and subjected to an incubation at 42°C for 2 min. Then, the mixture was supplemented with 5 μL of PrimeScript RT Enzyme Mix I, 20 μL of RT Primer Mix, 20 μL of 5× PrimeScript Buffer 2, and 5 μL of RNase-free distilled water (dH2O). After thorough mixing, the mixture was incubated at 37°C for 15 min, followed by a brief incubation at 85°C for 5 s. The cDNA products obtained were examined through PEC detection. The reagents and consumables required for each functional module were manually placed on the designed tray, after which the entire process was automatically completed by the robot.