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Particulate matter (PM2.5) as a potential SARS-CoV-2 carrier

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Tháng Hai 17, 2022
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Nội dung bài viết
  • Sampling location and indoor air sampling
  • Calibration of AiRBOXsense
  • Viral nucleic acid extraction
  • Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis

Sampling location and indoor air sampling

The layout and dimension of the wards are shown in Fig. 1. Each ward had different clusters of infected groups, as illustrated in Table 2 (in the main text) and Fig. 1. Each ward was occupied by one to eighteen COVID-19 patients. As a caveat, during the measurement in this study, hospital’s management staff has deployed three units of air purifiers at ward B, C, and D. During the air sampling measurement, air purifier (FANFIL AP510M, Aire-plus Technology, Singapore) was deployed at ~ 1 m distance in wards C and D, ~ 8 m in ward B, and no air purifier in single occupant room.

Figure 1
figure 1

The layout and dimension of the wards with instrumentation deployment a single room A b general ward B, c general ward C and d general ward D. (Note: The beds in the figure does not represent the actual number of beds in the wards).

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PM2.5 was sampled in a single-bed ward (31st March to 4th April 2020) and multiple bed wards (4th-29th April 2020) in a teaching hospital at Kuala Lumpur, respectively. Air sampling was conducted for 48 h during a 29 days sampling period using two types of instruments; an air quality sensor known as AiRBOXSense (AIRBOXSENSE V3.0, UKM Tech. Sdn Bhd, Malaysia)12,26 and a low volume sampler (LVS) (MINIVOL, AirMetrics, USA). Details of AiRBOXSense are described in26. Both instruments were operated side by side in wards occupied by SARS-CoV-2 positive patients. Instruments were treated using ultraviolet light for 20 min (UV) (UV-C 253.7 nm), further disinfected with 70% alcohol and calibrated before being translocated to the next wards. The same instruments were used to avoid variability during sampling.

AiRBOXSense was used to continuously measure PM2.5, while the LVS was used to determine the virus loading in PM2.5 trapped on filter paper (WHATMAN glass microfiber filters, Grade GF/F) with a tight specification of 0.6-0.8 μm particle retention and pure borosilicate glass structure, GF/F. A 5 L min−1 of air was drawn into the AiRBOXSense. While, the Minivol’s pump draws air at 5 L min−1 through a filter paper. The continuous concentration of PM2.5 was recorded and stored in secure digital card (SD card) in the AiRBOXSense. The data synchronously retrieved via THINGSPEAK (The MathWorks Inc, USA) cloud storage and analysed using MATLAB software (The MathWorks Inc, USA).

Each filter paper was collected after 48 h of sampling and stored in a sealed container and kept in − 80 °C laboratory freezer. The filter papers were extracted for viral load analysis using reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) approach.

Calibration of AiRBOXsense

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AiRBOXSense was calibrated according to26 1 day before each sampling. Calibration consists of setting a mathematical model describing the relationship between sensor data and reference instruments. The AiRBOXSense unit was calibrated in tandem with the GRIMM (as reference instrument) dust monitor model 1.108 (GRIMM Aerosol, Technik GmbH & Co. KG, Germany). The sensors measuring mass concentration were calibrated using GRIMM Aerosol, which was deployed at a clean area (laboratory) for lower concentration measurement and near to a car exhaust for high concentration measurement. The calibration equations are set by fitting a model during a calibration time interval when AiRBOXSense and GRIMM are co-located.

Viral nucleic acid extraction

Prior to viral nucleic acid extraction, the membrane filter was processed according to35 with slight modifications. The membrane was first divided into four parts and immersed in 1 mL sterile RNase-free water in separate tubes. Each part of the membrane was vortexed for 2 min in 30 s-intervals to release viral particles attached to the membrane. The tubes were then centrifuged at 500 rpm for 1 min to remove debris, and the supernatants were transferred into new microcentrifuge tubes for viral nucleic acid extraction. This process was repeated twice to ensure all virus particles were resuspended into the water. Subsequently, viral nucleic acid extraction was performed using a Viral Nucleic Acid Extraction Kit II (Geneaid Biotech Ltd., Taiwan) according to the manufacturer’s protocol. The purified nucleic acid containing the samples was then kept at − 80 °C for further analysis.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis

The primers and probes used in the detection of SARS-CoV-2 were 2019-nCoV_N1, and 2019-nCoV_N2 combined primer/probe mixes purchased from Integrated DNA Technology (IDT). The information on primers and probes were included in Table 1. Human RNase P primer was not included as a control in this analysis because this study was not conducted using specimen from human. RT-qPCR was carried out using a THUNDERBIRD One-step RT-qPCR kit (Toyobo Co., Ltd., Japan) according to the manufacturer’s protocol. The annealing temperature of the primers was set at 55 °C, as suggested by Centres for Disease Control and Prevention or CDC (2020)28. Detection of SARS-CoV-2 using the RT-qPCR approach with a BIORAD iQ5 Real-Time PCR machine (BIORAD, USA) as described by CDC (2020) with slight modifications. A standard curve was also generated using 2019-nCoV Positive Control (nCoVPC) with a series of tenfold dilutions from 2 × 105 to 2 copies/µL of the control template. The amplification efficiency and R2 value were recorded, and the standard curve was used to estimate the viral RNA of SARS-CoV-2 on the membrane.

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Table 1 Primers and probes sequences.
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