Extraction of Total Nucleic Acid from Environmental Samples for the Detection of Bacterial and Viral Targets
Dilip Abraham, Venkata Raghava Mohan, Mami Taniuchi, Nirmal kumar
Abstract
This procedure describes a modified extraction procedure to isolate both DNA and RNA from wastewater samples using the QIAamp Fast DNA Stool MiniKit. The sample undergoes a lysate preparation process and includes mechanical disruption (bead beating), removal of inhibitors, purification and elution of DNA and RNA using spin columns. Extrinsic controls SPC and MS2 are added to each sample during the lysate preparation to evaluate extraction and amplification efficiency. The extracted total nucleic acid (TNA) is then stored at -80°C for testing. To rule out contamination during the extraction process, a blank is also processed through the complete protocol each day extractions are performed. Briefly, the procedure comprises the following steps:
a. Lysis of and separation of impurities from sample pellets in InhibitEX buffer.
b. Purification of DNA and RNA on QIAamp Mini spin columns.
Before start
Extraction control preparation
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DNA control Before first use of the sample process control (SPC), prepare a 1/10 dilution of the control DNA in nuclease free water and store them at -70 °C as small aliquots to avoid freeze thaw. Add 1 µL of 1/10 diluted SPC into particle pellet.
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RNA control Prepare a 1/10 dilution of the MS2 extraction control in nuclease free water and store them at -70 °C as small aliquots to avoid freeze thaw. Add 1 µL of 1/10 diluted MS2 into particle pellet.
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For Nanotrap® samples, start the TNA extraction from step 9 in the protocol referenced below. Wastewater grab sample processing with Nanotrap® Microbiome A particles (40 ml)
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For Moore swab samples, the pellet consists of membrane filter strips as described in the protocol below. Environmental sampling using Moore swabs
Steps
Total Nucleic Acid extraction (TNA)
Kit used
For samples processed by Nanotrap® A microbiome particles: Follow the steps described in the referenced protocol as described in the guidelines above.
For Moore swab samples: Add the cut membrane filter strips to 2ml Screw Cap Micro Tubes containing ~ 370mg (one 2 ml eppendorf tube capful) of acid-washed glass beads.
Add both extraction controls to the tube, and then add 700µL Inhibitex buffer.
For pellets derived from other wastewater sample concentration methods: Resuspend the pellet in 3mL of sterile PBS, and add 1ml of this suspension to 2ml Screw Cap Micro Tubes containing ~ 370mg (one 2 ml eppendorf tube capful) of acid-washed glass beads.
Add both extraction controls to the tube, and then add 700µL Inhibitex buffer.
Include one extra tube for extraction blank (bead + InhibitEX buffer).
Shake the tubes on a bead beater at maximum speed for 0h 2m 0s
Incubate the suspensions for 0h 5m 0s at 95°C
Vortex for 0h 0m 15s.
Centrifuge the samples at full speed (approximately 20,000g) for 0h 1m 0s to pellet the particles.
Pipette 25µL Proteinase K into a new 2mL microcentrifuge tube.
Transfer ~600µL of supernatant from step 9 into the 2 ml tube containing proteinase K.
Add 600µL of Buffer AL to the mix.
Vortex for 0h 0m 15s . Mix thoroughly to form a homogeneous solution.
Incubate at 70°C for 0h 10m 0s . Centrifuge briefly to remove drops from the inside of the tube lid.
Add 600 μl of ethanol (96–100%) to the lysate and mix by vortexing. Centrifuge briefly to remove drops from the inside of the tube lid.
Add 600µL of lysate from step 15 onto a QIAamp spin column (in a 2 ml collection tube) without moistening the rim. Close and label the cap.
Centrifuge at full speed for 0h 1m 0s . Retain the spin column and discard the flow-through and collection tube.
Place the QIAamp spin column in a new 2mL collection tube and repeat step 16 - 17 twice until all lysate (~1800µL ) is passed through the spin column.
Place the QIAamp spin column in a new 2mL collection tube and add 500µL Buffer AW1.
Centrifuge at full speed for 0h 1m 0s. Retain the spin column and discard the flow-through and collection tube.
Place the QIAamp spin column in a new 2mL collection tube and add 500µL Buffer AW2.
Centrifuge at full speed for 0h 3m 0s . Retain the spin column and discard the flow-through and collection tube.
Place the QIAamp spin column in a new 2mL collection tube and centrifuge at full speed for 0h 3m 0s to eliminate the chance of possible Buffer AW2 carryover.
Place the QIAamp spin column in a new 1.75mL microcentrifuge tube, add 100µL of Buffer ATE directly onto the QIAamp membrane.
Incubate at Room temperature for 1 - 3 min.
Centrifuge at full speed for 0h 1m 0s to elute the TNA.
Discard the spin column and save the filtrate containing TNA.
Aliquot 50µL of extracted TNA into 1.75mL screw cap tube and store at -80°C for long term storage.
The remaining 50µL of eluate is stored at -80°C and use for further analysis.
