An uptick in recent epidemics, such as Zika and Ebola virus, have highlighted the need for a more coordinated and nimble response on a global scale. To accomplish this we have designed an innovative, scalable, intra-modal laboratory unit to be used in resource-constrained areas worldwide during such outbreaks. Here we present our intra-modal, expandable, mobile shipping container laboratories.
And in the following paper, we present our BSL-2 and BSL-3 approach to respiratory influenza virus diagnosis. Rapid influenza virus diagnostics by RCT-PCR. Before preparing to enter the installed laboratory unit, be aware that all BSL-2 safety requirements must be accounted for.
This includes dressing with proper personal protective equipment, washing hands, wearing gloves, and decontaminating any workspaces you plan to use. Note, if you use bleach to decontaminate your workspace and supplies, you must also use 70%ethanol to clean all areas exposed to bleach, as bleach can mix with other chemicals in the workspace to create toxic fumes. Dispose all bleach products into their own designated waste bin.
As sample swabs are taken from patients, they are transported to the laboratory facility from the field or clinic and passed to the lab technician via the pass-through window. This window cannot be opened from both sides. In the BSL-2 pass-through window, the external surface of tubes containing samples must be completely disinfected with bleach and left alone to sit for two minutes.
Open the pass-through window and collect the samples to be registered. Wipe down any samples that have remnants of bleach, and wipe down the interior of the pass-through window with bleach, followed by 70%ethanol solution. Add bar codes to each sample tube, and your four empty tubes designated for aliquots.
Move your samples to the vent hood. Scan the barcode on each tube and make sure that the proper sample identification information appears on the tablet-based system or laptop screen. Complete the registration process.
Sample aliquot. Once test tubes have been labeled, use a certified and decontaminated class II biosafety cabinet to take aliquots of samples. When taking aliquots be sure to use fresh sterile or disposable pipettes for each sample and discard them into biohazardous waste containers in order to avoid cross contamination.
Make sure every tube is tightly sealed and closed. Utilize one aliquot per specimen for immediate extraction. Store any other aliquots in the freezer for future use.
Before moving to the workstation, clean all workspace surfaces and equipment with bleach followed by 70%ethanol solution. Once your barcoded PCR samples have been aliquoted move them to a workstation designated for extraction. Prepare your buffer AVL carrier RNA mixture for the lysis step.
The sample and lysis buffer master mix should be kept at room temperature. Label a 1.5-milliliter centrifuge tube. Set the pipette tip to 560 microliters.
Apply a clean pipette tip. Add a lysis buffer to each tube. Discard the tip in the waste container.
Next, take your PCR aliquot sample. Place it into the prepared lysis buffer tube designated for aliquot one, and repeat this for any additional samples. Discard old pipette tip and apply a clean pipette tip.
Apply a clean pipette tip. Add 140 microliters of a buffer to the control tube. Close each tube securely.
Pulse-vortex for 15 seconds. Repeat with any additional aliquots or samples. Microcentrifuge each sample for five seconds.
Incubate at room temperature for 10 minutes. After 10 minutes briefly centrifuge your tubes to remove any drops from the inside of each tube's lid. Add 560 microliters of ethanol the sample one.
Change your pipette tip. Repeat with any remaining or additional samples. Close each sample tube securely.
Pulse-vortex each sample for 15 seconds. Microcentrifuge the samples for five seconds. Obtain a clean two-milliliter collection tube.
Add the spin columns. Label them to match your samples. Transfer 630 microliters of the sample to match the column accordingly.
Secure your caps. Move to the centrifuge. Evenly distribute your samples in the centrifuge.
Centrifuge at 6000 g's for one minute to remove the lysis buffer. Return to workstation. Replace your collection tubes.
Add the remaining lysis buffer and repeat the centrifugation step. Discard your original aliquot sample tubes, after which, dispose the eluate and wash the spin column with the buffer AW1. Repeat this procedure with every sample or any additional samples.
Secure the caps on each sample and centrifuge at six g's. Repeat with the second wash buffer AW2 at 20 g's. Finally, place the column into a clean 1.5-milliliter tube.
Open the column and add 60 microliters of buffer AVE. Incubate at room temperature for one minute and centrifuge at six g's for one minute. The sample is now ready for PCR analysis.
Under BSL-3 conditions experimental protocol will stay the same, but safety measures will take precedent above anything else. Before entering the BSL-3 lab look through the transparent window to be sure negative pressure has been established in the glovebox unit. It will be evident that negative pressure has been established when the ball in the wall is visible.
Once negative pressure has been established open the door and enter the unit. Immediately wash your hands and then proceed to your PPE checklist. Proceed to putting on the PPE in the following order:under gloves, gown, shoe covers, mask, face shield, second pair of gloves.
Turn on the machine and allow the pressure in the glovebox to stabilize. Use a bleach spray solution to decontaminate all areas and supplies you plan to use inside and outside the glovebox. Dispose of any bleach waste products into a bleach-only container.
Use 70%ethanol solution to clean over any areas where bleach is has been used. Transfer your samples via the pass-through window. Samples should have been sprayed with bleach solution and left alone for at least one minute in the pass-through before receiving them inside of the BSL-3 unit.
Next, receive your samples inside of the BSL-3 unit and clean them thoroughly before proceeding to the registering and labeling step. Once samples are registered and tubes have been labeled, place the samples into the glovebox via the airlock tray. Do not open both doors at once.
Open and close each door in two different steps for safety precautions. Once samples have been safely moved to the glovebox interior, follow the same steps as previously described for creating sample aliquots in the glovebox. Once samples are aliquoted move the samples into the airtight container.
Spray bleach solution within the glovebox to decontaminate tubes and all other surfaces. Wait five minutes and then apply 70%ethanol solution to wash the bleach. After thorough decontamination move only the samples needed for PCR analysis for the lysis step back into the glovebox.
In the glovebox perform only the lysis step of the extraction procedure. Once the cells have been lysed, tightly seal the caps on each sample and place them into the pressurized airlock passage. Decontaminate the glovebox again with bleach followed by 70%ethanol solution.
Move the samples out of the glovebox and transfer them to the workstation for the remaining steps of the extraction procedure. After extraction transfer your samples to the pass-through window for PCR analysis. After samples have been removed and transferred, decontaminate your laboratory workspace outside of the glovebox.
Before removing your PPE, wait until air circulation in the unit has safely reached a proper number of filtering cycles before you begin to remove your PPE. Immediately after removing and disposing of all PPE into the PPE waste container, proceed to washing your hands in the lab sink with soap and water before exiting the unit. PCR amplification and detection.
Remove the extracted RNA samples from the pass-through window. Prepare a master mix in a 1.5-milliliter tube with the following components for each targeted assay:water, primers and probes, attack mixture, and 2X buffer. Vortex and spin your master mix, after which, aliquot the master mix into each of the strip tubes.
Return the PCR kit to storage at the recommended temperatures once the master mix has been prepared. Add the samples to each of the strip tubes using a separate tip between each strip tube. Spin the samples at 1, 500 rpm for one minute, after which samples are ready to be loaded onto the real-time PCR unit.
Once samples are loaded onto the PCR instrument, it takes approximately 90 minutes to complete one run. Real-time PCR assay is a sensitive method for robust detection of the virus. This can be seen in the table labeled:PCR Analyzed Results.
Before collecting your results and leaving the lab, remove PPE and adequately decontaminate each workstation in preparation for the next diagnostic test. Representative results. The laboratory workflow is represented in the diagram which emphasizes personal protection.
The rapid diagnostic test of influenza is accomplished via RT-PCR analysis. The procedure consists of four main steps and individual workspaces are assigned for each stage of the protocol. The goal of this project is to validate a novel modular rapidly-deployable laboratory facility and we have successfully done this, as well as develop training programs for laboratory personnel working in low-resource environments during epidemics and outbreaks.
