This method can help answer key questions about the type, level, and distribution of viruses in bioaerosols that are important for public and veterinary health. The main advantages of this method are its improvement on the sensitivity of molecular detection of viruses from bioaerosols, its field amenability, and its ease of use. Create suspensions of the experimental concentrations of viral particles of interest in 100 milliliter volumes of PBS within the glass vessels of a six-jet collision nebulizer.
To set up the bioaerosol chamber, preload two liquid impingers with 20 milliliters of 0.01 molar PBS followed by the addition of 0.5 grams of anion exchange resin to a single impinger. Assemble the impinger. Install the suspensions within the chamber.
Use clamp stands to position liquid impingers in parallel inside the bioaerosol chamber with the aerosol sampler inlets facing the nebulizer. Place a direct read instrument near the liquid impingers to monitor the environmental variables and place an additional direct read aerosol monitor near the liquid impingers to measure the mass concentration of the bioaerosol. Then, run the axial fans in the corners of the bioaerosol chamber to facilitate mixing of the aerosol.
When the chamber is ready, start the instruments for measuring the variables within the bioaerosol chamber. Use a primary flow standard to calibrate the system to maintain the consistency between the sampling volumes. Seal the chamber for a 10 minute purge with HEPA filtered air.
Deliver filtered, dried air to the nebulizer at 6.9 kilopascals of pressure to generate a target concentration of viral bioaerosols. When the target mass concentration of the aerosol has been reached, stop the nebulization. Then, use a HEPA filtered line located near the nebulizer to supply diluted air, taking care to maintain a constant pressure within the chamber and actively sample the bioaerosol chamber atmosphere for 40 minutes to achieve a sampling volume of 500 liters per sample.
Calibration of the instrument is critical for determining the concentration of air contaminants. The sampling train should be calibrated before and after each sampling event. Pre and post calibrations should be within 5%of one another.
Turn off the pumps at the end of the sampling. To isolate nucleic acids directly from the anion exchange resin, transfer all of the anion exchange resin containing liquid from the liquid impingers into individual, sterile 50 milliliter conical tubes. After allowing the resin to settle, slowly decant the liquid sample from the anion exchange resin, using a one milliliter pipette tip to remove any remaining liquid from the resin.
Next, add 560 microliters of viral lysis buffer containing carrier RNA from a viral RNA isolation kit directly to the anion exchange resin, for a 10 minute incubation at room temperature with periodic mixing. At the end of the incubation, use a one milliliter pipette tip to transfer the viral lysate to a sterile 1.5 milliliter conical tube and proceed with the RNA isolation according to the manufacturer's instructions. Then, perform quantitative real-time PCR for detection of MS2 bacteriaphage and influenza A and B viruses using five microliters of nucleic acid eluent per sample.
As shown in these representative qRT-PCR amplification curves, the detection of Type A influenza viruses can be achieved up to 3.26 cycles sooner when anion exchange resin is used, compared to direct testing of the impingement liquid. In a qRT-PCR that is 100%efficient, a gain of one threshold cycle corresponds to a two-fold increase in target concentration, thus the representative results indicate a 9.58 fold improvement in detection sensitivity. Once mastered, this technique can be completed in less than three hours if performed properly.
While attempting this procedure, it's important to remember that when air sampling is conducted in the field, parameters such as relative humidity and temperature may affect collection efficiencies and that collection buffers may have to be optimized for different viruses. Following this procedure, other culture based methods can be utilized to answer additional questions about viral infectivity. After its development, this technique has paved the way for researchers and other professionals in fields of agricultural and public health, to explore alternative methods for viral capture and concentration from bioaerosols for risk assessment in viral disease surveillance.
After watching this video, you should have a good understanding of how to capture and concentrate viruses from bioaerosols using modified liquid impingers, extract nucleic acids directly from the anion exchange resin and detect viruses using qRT-PCR. Don't forget that working with viral pathogens can be extremely hazardous and precautions such as using personal protective equipment and bio-safety level two or higher should always be taken when performing this procedure.
