TITLE: Sewage sample extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure
AUTHORS: Dilip Abraham, Venkata Raghava Mohan, gkang 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This procedure describes a modified extraction procedure to isolate both DNA and RNA from pellet obtained from secondary concentration from BMFS samples using the QIAmp 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 PhHV (Phocine Herpesvirus) 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.</div></div>

[STEPS]
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Mix InhibitEX Buffer thoroughly by shaking before use. If a precipitate has formed, Incubate at - for ~ or until all precipitate has fully dissolved.
37 °C
70 °C
InhibitEX buffer is a lysis buffer, designed to break open the cells. Important components of the lysis buffer are detergents, enzymes, and salts. Sometimes, the salts form precipitates in the solution and fall to the bottom, so when you pipet some of the solution to use in DNA extraction, it will be missing the salts and might not work properly and might not break open the cells.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Prepare fresh each day: Add PhHV and MS2 working solutions to InhibitEX buffer: prepare only enough for N+1 samples to be processed that day:  InhibitEX buffer + PhHV+ MS2 is needed per sample (e.g. For 10 samples  InhibitEX+  PhHV+MS2), mix well.
1 mL
1 µl
1 µl
11 mL
11 µl
11 µl
PhHV (Phocine Herpes Virus) is a double-stranded DNA virus and MS2 is a single stranded RNA virus.They are used as internal controls to ensure the DNA extraction and PCR worked properly. We add them to the DNA extraction buffers in a known quantity and if they are not detected in the final sample at a specific level, we know that either 1) some kind of problem occurred in DNA extraction or 2) there were inhibitors in the PCR. During analysis we might consider removing these samples or adjusting the results in some way to account for the inhibition.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
If the beads have not been pre-aliquoted to empty tubes: Add : ~ (one eppendorf tube capful) of Sigma beads to the aliquot of stool (or blank tube).
370 mg
Some bacterial cells and parasite cells are difficult to break open with just chemical lysis buffers. So, we also use mechanical lysis (using a physical thing – beads – to break open the cell).
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Add  of the InhibitEX/PhHV/MS2 solution to the first skim milk pellet in  conical then resuspend the pellet using a  serological pipette tip then resuspend the 2nd pellet with the suspension from the first tube and continue until all 6 pellets are suspended and combined (or blank).
1 mL
50 mL
2 mL
DNA-degrading substances and PCR inhibitors present in the stool sample are separated from the DNA by the InhibitEX buffer.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Add the combined suspension into a  screw cap tube with the glass beads.
2 mL
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Vortex for .
This step is just to ensure that the lysis buffer is mixed well and all beads are moving (not stuck in the bottom of the tube) so that in the next step the cells will be agitated fully with both the chemical and mechanical lysis.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Bead beat at maximum speed for - .
This step is mostly for mechanical lysis of cells with the beads.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Incubate the suspension for  at .
95 °C
Human cells lyse at room temperature, but many bacterial and parasite cells are stable at this temperature, even in the presence of chemical lysis buffers. So, here we heat the solution to assist in the lysis of the cells that might have survived the chemical and mechanical lysis in the previous steps.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Vortex for  then centrifuge sample at full speed (approximately ) for  to pellet the stool particles.
Centrifuge: 20000 34
We centrifuge to pull down all large components remaining in the sample matrix (like food particles remaining in the stool, cell wall debris, or other things not suspended in the liquid portion of the buffer).
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Pipet  proteinase K into a new  microcentrifuge tube, (snap caps acceptable).
25 µl
2 mL
Proteinase K digests and degrades proteins during incubation at  .
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Pipet  supernatant from step 8 into the  microcentrifuge tube containing proteinase K.
600 µl
2 mL
Note: Do not transfer any solid material, if necessary centrifuge sample again.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Add  Buffer AL.
600 µl
Note: Do not add proteinase K directly to Buffer AL.Sodium (Na+) ions in the buffer neutralize the negative charges on the DNA molecules, which makes them more stable and less water soluble. By optimizing these salt and pH conditions with the AL buffer, we ensure that any remaining digested proteins and other impurities (which can inhibit PCR) are not retained.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Vortex for , Mix thoroughly to form a homogeneous solution.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Incubate at  for . Centrifuge briefly to remove drops from the inside of the tube lid.
70 °C
Proteinase K is working during this step to degrade proteins.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Add  of ethanol (96–100%) to the lysate, and mix by vortexing. Centrifuge briefly to remove drops from the inside of the tube lid.
600 µl
DNA is not soluble in ethanol so it will precipitate out of the solution and onto the spin column membrane in the next step.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Label QIAamp spin column lid and place in a  collection tube. Carefully apply  of the lysate from step 14 to the QIAamp spin column without moistening the rim. Close the cap and centrifuge at full speed for .
2 mL
600 µl
The special membrane in the bottom of the spin column will bind the DNA and let other dissolved components pass through into the filtrate that comes out the bottom (which we then discard).
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Retain QIAamp spin column, place the column in a new  collection tube, and discard the collection tube containing the filtrate.
2 mL
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Repeat 2 more times to use all of the lysate: carefully apply an additional  of the lysate from step 14 to the QIAamp spin column without moistening the rim. Close the cap and centrifuge at full speed for . Retain the column. Discard the collection tube and filtrate. Repeat until all of the lysate ( ~ in total) has been through the spin column.
600 µl
1800 µl
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Place the spin column in a new collection tube.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Open the QIAamp spin column and add  Buffer AW1. Close the cap and centrifuge at full speed for . Place the QIAamp spin column in a new  collection tube, and discard the collection tube containing the filtrate.
500 µl
2 mL
We wash the DNA that is bound to the membrane with Buffer AW1 (Wash Buffer1). This ensures we remove any residual impurities.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Open the QIAamp spin column and add  Buffer AW2. Close the cap and centrifuge at full speed for . Retain QIAamp spin column, discard the collection tube containing the filtrate.
500 µl
Buffer AW2 removes another type of impurity (I don’t know the specific things that each wash buffer targets!).
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
To eliminate the chance of possible Buffer AW2 carryover place the QIAamp spin column in a new  collection tube and discard the old collection tube with filtrate. Centrifuge at full speed for .
2 mL
Note: Residual Buffer AW2 in the eluate may cause problems in downstream applications. An extra centrifugation step ensures that we remove all excess Buffer AW2.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Elute the total nucleic acid: Transfer the QIAamp spin column into a labeled microcentrifuge tube. Open the QIAamp spin column and pipet  Buffer ATE directly onto the QIAamp membrane. Close the cap and incubate for 1- at , then centrifuge at full speed for  to elute the TNA.
200 µl
70 Room temperature
The ATE buffer re-dissolves the DNA from the membrane back into solution. The incubation for  -  is important to allow enough time for the DNA to unbind from the membrane/ dissolve into the solution. During centrifugation, the DNA passes thru the filter into the final collection tube with the buffer.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Save the filtrate: this is the TNA. Discard the column.
?. [Extraction of total nucleic acid from secondary concentration pellet using a modified QIAamp Fast DNA Stool Mini Kit procedure]
Prepare two tubes for aliquot storage. Pipet  of the extracted TNA into each tube. Store at  to .
100 µl
-70 °C
-80 °C
?. [Appendix 1 Preparation and Storage of MS2/PhHV mix]
Quickly spin the tube containing lyophilized MS2/PhHV mix to bring the pellet to the bottom.
MS2 and PhHV are incorporated during extraction as extrinsic controls in order to evaluate the extraction and amplification efficiency of RNA and DNA. This protocol describes the preparation and storage of MS2/PhHV mix.Material1. Lyophilized MS2/PhHV mix, store at   until use. Reconstitute one vial at a time.2. STD Diluent, store at  .
?. [Appendix 1 Preparation and Storage of MS2/PhHV mix]
Add  STD diluent to each vial, mix well.
50 µl
?. [Appendix 1 Preparation and Storage of MS2/PhHV mix]
Use  per sample extraction follow the extraction SOP. Store the leftover at .
1 µl
-80 °C
Don’t use the reconstituted mix that has been frozen-thawed more than two times.