Structure of the electrostatic samplers
Electrostatic precipitation is a method of charging and collecting suspended particles by corona discharge. The smaller the radius of curvature of an electrode, the lower the voltage required to generate corona discharge. A bundle of 100 metallic fibers with 12 µm diameter was used as the discharge electrode21. About 2 mL of Buffer AVL (QIAGEN, Germany) supplied in the vial, which was used as the collection electrode. The buffer is a viral lysis solution used for purifying viral nucleic acids. Using the buffer as the collection electrode, we were able to reduce the risk of infection to workers. Figure 3 shows the schematic diagram of the sampler. A metal tube with 20 mm diameter was inserted into the center of a vial with 40 mm diameter. 11 bundles for corona discharge were attached to the tip of the tube. The gap between the discharge electrode and the surface of the buffer AVL was ଁxed to 10 mm. The applied voltage was DC-6 kV and the discharge current was -80 µA. The sampling air flow rate was 40 L/min. The air flowing in from the central metal tube was turned back in the vial and exhausted to the surrounding area. Meanwhile, the charged aerosol is collected into the buffer by electrostatic force.
Collection efficiency of the electrostatic samplers
Incense smoke was mixed with air to form sample gas and used in a room with a volume of 100 m3. This was conducted to increase the number of airborne particles with 300 nm to 5000 nm of diameter in the indoor air. The sample gas was passed through the electrostatic sampler to measure the collection efficiency. A Fast Mobility Particle Sizer (FMPS; TSI, USA) and an optical particle counter Model 3888 (Kanomax, Japan) were used. FMPS can measure particles from 5.6 nm to 560 nm, and the particle counter can measure particles from 0.3 µm to 5.0 µm. The collection efficiency was calculated using the ratio of particle-count with the high voltage (HV) turned ON and OFF.
Possibility of damage to nucleic acids by corona discharge
To confirm the effect on nucleic acids, bacteriophage MS2 was exposed to the discharge, and analyzed using RT-qPCR. Bacteriophage MS2 (NBRC 102619) and Escherichia coli (NBRC 13965) were mixed and incubated overnight at 35℃ in liquid LB medium. 4 mL of SM buffer and 40 µL of chloroform were added into the mixture and allowed to stand at room temperature for 30 minutes. The mixture was filtered through a syringe filter with a pore size of 0.22 µm. Bacteriophage MS2 suspension with concentration of 5 x 107 PFU/µL was obtained.
1 µL of bacteriophage MS2 suspension was added to 2.9 mL of solution (1.12 mL of Buffer AVL and 1.78 mL of DW, DW: deionized water). DW was added to prevent salt deposition caused by evaporation. The solution was supplied in a vial and was used as the collection electrode. Samples were prepared by applying corona discharge and air flow or air flow only for 90 min.
Bacteriophage MS2 RNA extraction and RT-qPCR detection
RNA was extracted from each sample using the QIAamp Viral RNA mini kit (QIAGEN, Germany) following the protocol. The presence of bacteriophage MS2 RNA was detected using One Step Prime Script III RT-qPCR Mix (TAKARA BIO, Japan) and primer-probe sets targeting of the coat protein. The sequences of the primers and probe are 5‘-CGTTCACAGGCTTACAAAGTAACCT-3’, 5’-CCAACAGTCTGGGTTGCCAC-3’, and FAT-AGAATCGCAAATACACCATCAAAGTCGAGGT-TAMRA, respectively22. Reaction mixtures contained 10 µL of One Step Prime Script III RT-qPCR Mix, 1 µL of each 0.5 µM primer, 1 µL of 0.5 µM probe, 6.0 µL of RNase Free H2O, and 1 µL of extracted RNA. The PCR conditions were 52°C for 5 min, 95°C for 10 sec and 45 cycles of 95°C for 10 sec and 60°C for 30 sec. The PCR device was used Mygo Mini S Real Time PCR (IT-IS Life Science, UK).
Sampling of indoor air
The sampling was conducted at a food court of a commercial facility, a passage of a clouded train station, and two different offices (A and B). The food court of the commercial facility was about 2000 m3 in size, and about 300 people were eating and talking without masks. The passage of the clouded train station was about 1500m3 in size, and about 100 people were walking through it at any given time. Office A was 850 m3 in size, and there were 60 people in the room. Office B was 340 m3 in size, and there were 30 people in the room.
Two types of prototype devices (A and B) were used in the demonstration test. Device A consists of one sampler with the sampling air flow rate of 40 L/min, and device B consists of three samplers in parallel with the sampling air flow rate of 120 L/min. The size of both devices was 300 mm (length), 300 mm (width), and 150 mm (height), and they were powered by a battery with 12 V output. The buffers for each sampler were 2.9 mL (1.12 mL of Buffer AVL and 1.78 mL of DW). DW was added to prevent salt deposition caused by evaporation. Sampling time was 90 minutes. The volume of solution after sampling was 1.4 mL for device A and 4.2 mL (1.4 mL x 3) for device B.
Viral RNA extraction and RT-qPCR detection
RNA was extracted from each sample using the QIAamp Viral RNA mini kit (QIAGEN, Germany) following the protocol. The extracted solution was 60 µL. The presence of SARS-CoV-2 RNA was detected targeting the N and open reading frame (ORF) gene regions.
In the N region, the kit used for detection is One Step Prime Script III RT-qPCR Mix (TAKARA BIO, Japan), Primer/Probe Set (TAKARA BIO, Japan), and Positive Control RNA Mix (2019-nCov) as positive control. Reaction mixtures of N1 set contained 10 µL of One Step Prime Script III RT-qPCR Mix, 4.0 µL of 5 x N_Sarbeco_Primer/Probe mix, 1.0 µL of RNase Free H2O, and 5 µL of extracted RNA. Total volume is 20 µL. Reaction mixtures of N2 set contained 10 µL of One Step Prime Script III RT-qPCR Mix, 4.0 µL of 5 x NIID_2019_nCOV_N_Primer/Prove mix, 1.0 µL of RNase Free H2O, and 5 µL of extracted RNA. Total volume is 20 µL. The PCR conditions were 52℃ for 5 min, 95℃ for 10 sec and 45 cycles of 95℃ for 10 sec and 60℃ for 30 s. The PCR device was used Mygo Mini S Real Time PCR (IT-IS Life Science).
In the ORF region, the kit used for detection is 2019-nCoV Detection Fluorescence RT-qPCR Kit (Sysmex, Japan). Reaction mixtures contained 18.5 µL of 2019-nCoV Reaction Mix, 1.5 µL of 2019-nCoV Enzyme Mix and 10 µL of extracted RNA. Total volume is 20 µL. The PCR conditions were 50℃ for 20 min, 95℃ for 10 min and 45 cycles of 95℃ for 15 sec and 60℃ for 30 sec. The PCR device is the same as above.
A calibration curve was prepared using the positive control for the N region. For the ORF region, a calibration curve could not be drawn due to the low concentration of the commercial positive control. The positive control was adjusted by 10-fold dilution from 1.0 x 100 to 1.0 x 103 copies/µL, and calibration curves were drawn for the N 1 and N 2 regions using the PCR conditions described above. The RT-qPCR data were quantitatively analyzed using the calibration curve and the suspended concentration per 1 m3 of aspirated air was calculated.