TITLE: Extraction of Total Nucleic Acid from Environmental Samples for the Detection of Bacterial and Viral Targets
AUTHORS: Dilip Abraham, Venkata Raghava Mohan, Mami Taniuchi, Nirmal kumar

[DESCRIPTION]
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

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. 

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. 

For Nanotrap‱ samples, start the TNA extraction from step 9 in the protocol referenced below.
 

For Moore swab samples, the pellet consists of membrane filter strips as described in the protocol below.
 


[GUIDELINES]
The pellet obtained after sample concentration with Nanotrap‱ A particles should not be frozen. Extraction should be carried out the same day.

[STEPS]
SECTION: Total Nucleic Acid extraction (TNA)
1.   
Kit used
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3. For Moore swab samples: Add the cut membrane filter strips to 2ml Screw Cap Micro Tubes containing   ~ 370 mg (one 2 ml eppendorf tube capful) of acid-washed glass beads.

SECTION: Total Nucleic Acid extraction (TNA)
5. Include one extra tube for extraction blank (bead + InhibitEX buffer).
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6. Shake the tubes on a bead beater at maximum speed for 2 min 
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7. Incubate the suspensions for 5 min  at 95 °C 
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8. Vortex for 15 s.
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10. Pipette 25 µL  Proteinase K into a new 2 mL microcentrifuge tube. 
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11. Transfer ~600 µL of supernatant from step 9 into the 2 ml tube containing proteinase K.
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12. Add 600 µL  of Buffer AL to the mix.
 

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13. Vortex for 15 s . Mix thoroughly to form a  homogeneous solution.

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14. Incubate at 70 °C for 10 min . Centrifuge briefly to remove drops from the inside of the tube lid.
SECTION: Total Nucleic Acid extraction (TNA)
16. Add 600 µL 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. 
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18. Place the QIAamp spin column in a new 2 mL  collection tube and repeat step 16 - 17 twice until all lysate (~1800 µL ) is passed through the spin column.
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19. Place  the QIAamp spin column in a new 2 mL collection tube and add 500 µL Buffer AW1. 
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21. Place the QIAamp spin column in a new 2 mL collection tube and add 500 µL Buffer AW2.

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24. Place the QIAamp spin column in a new 1.75 mL microcentrifuge tube, add 100 µL of Buffer ATE directly onto the QIAamp membrane.
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23. Place the QIAamp spin column in a new 2 mL collection tube and centrifuge at full speed for 3 min to eliminate the chance of possible Buffer AW2 carryover.
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27. Discard the spin column and save the filtrate containing TNA.
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28. Aliquot 50 µL of extracted TNA into 1.75 mL screw cap tube and store at -80 °C  for long term storage.

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29. The remaining 50 µL of eluate is stored at -80 °C  and use for further analysis.
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15. 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.
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2. For samples processed by Nanotrap‱ A microbiome particles: Follow the steps described in the referenced protocol as described in the guidelines above.
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3.1.  Add both extraction controls to the tube, and then add 700 µL  Inhibitex buffer.
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9. Centrifuge the samples at full speed (approximately 20,000g) for 1 min to pellet the particles.
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17. Centrifuge at full speed for 1 min . Retain the spin column and discard the flow-through and collection tube.
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20. Centrifuge at full speed for 1 min. Retain the spin column and discard the flow-through and collection tube.
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22. Centrifuge at full speed for 3 min . Retain the spin column and discard the flow-through and collection tube.
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25.  Incubate at Room temperature for 1 - 3 min.
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26.  Centrifuge at full speed for 1 min  to elute the TNA.
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4. For pellets derived from other wastewater sample concentration methods: Resuspend the pellet in 3 mL of sterile PBS, and add 1ml of this suspension to 2ml Screw Cap Micro Tubes containing   ~ 370 mg (one 2 ml eppendorf tube capful) of acid-washed glass beads.

SECTION: Total Nucleic Acid extraction (TNA)
4.1.  Add both extraction controls to the tube, and then add 700 µL  Inhibitex buffer.