May 08, 2026
Sample processing, nucleic acid extraction and screening (q/d-PCR) protocol for environmental surveillance of Influenza A virus from wetland and poultry settings
- Bhumika Prajapati1,
- Rameshchandra Pandit1,
- Amrutlal Patel1,
- Shewane Bishnoi1,
- Abhinandan Patnaik1,
- Tejas Shah1,
- Kalpesh katudia1,
- Roselin Neihsial1,
- Jinal Thakor1,
- Jill Gada1,
- Rajvi Der1,
- Parth Pandit1,
- Kunjan Panchal1,
- Bhagirath Dave1,
- Harshal Purohit1,
- Snehal Bagatharia1,
- Amit Kanani2,
- Dhwani Pandya1,
- Madhvi Joshi1,
- Chaitanya Joshi1,
- Debashrita Mittra1
- 1Gujarat Biotechnology Research Centre, Dept. of Science and Technology, GoG, Gandhinagar, Gujarat, India;
- 2Deputy Director , Directorate of Animal Husbandry , Government of Gujarat
Protocol Citation: Bhumika Prajapati, Rameshchandra Pandit, Amrutlal Patel, Shewane Bishnoi, Abhinandan Patnaik, Tejas Shah, Kalpesh katudia, Roselin Neihsial, Jinal Thakor, Jill Gada, Rajvi Der, Parth Pandit, Kunjan Panchal, Bhagirath Dave, Harshal Purohit, Snehal Bagatharia, Amit Kanani, Dhwani Pandya, Madhvi Joshi, Chaitanya Joshi, Debashrita Mittra 2026. Sample processing, nucleic acid extraction and screening (q/d-PCR) protocol for environmental surveillance of Influenza A virus from wetland and poultry settings. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoem9xl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 06, 2026
Last Modified: May 08, 2026
Protocol Integer ID: 316416
Keywords: viral rna extraction, precipitation viral enrichment, air from poultry farm, poultry drinking water, air from wetland, nucleic acid extraction, extracted rna, influenza, viral enrichment, wetland surface water, wetland water, including wetland water, wetland water sediment, poultry settings this protocol, poultry setting, virus, wetland, based silica column purification method, poultry farm, cage swab, quantitative pcr, silica column purification method, bird dropping, molted feather, feather, bird
Funders Acknowledgements:
Gates Foundation
Grant ID: INV-064425
Abstract
This protocol describes a standard process for surveillance of Influenza A virus (IAV) from different environmental samples including wetland water, water sediment, molted feathers, bird droppings, and air from wetlands. While drinking water, bird’s cage swab, bird droppings, litter, molted feathers, and air from poultry farms. The wetland surface water, poultry drinking water, wetland water sediment and bird’s cage swab are processed by polyethylene glycol (PEG)–Sodium chloride (NaCl) precipitation viral enrichment, followed by viral RNA extraction using modified TRIzol isolation combined with kit-based silica column purification method. Molted feathers, bird droppings, litter, and air samples collected in VTMs are directly subjected to viral RNA extraction using automated magnetic bead–based extraction protocol. Extracted RNA can be screened for the presence of IAV using real-time quantitative PCR (qPCR) and digital PCR (dPCR).
Materials
- 50 or 100 mL centrifuge tubes
- Nuclease free micro-centrifuge tubes (1.5 mL)
- 200 µL, 20 µL and 10 µL filter micropipette tips
- Micropipettes
- Polyethylene glycol (PEG-8000), HIMEDIA
- Sodium Chloride (NaCl), Hi-AR% HIMEDIA
- Nuclease free water
- High speed refrigerated centrifuge
- Kimwipes/tissue paper
- Vortex mixer
(A) Sample processing and virus enrichment
The aim of this step is to concentrate virus particles from environmental samples using polyethylene glycol (PEG) and sodium chloride (NaCl). This process enhances the detection and further analysis of viruses for downstream applications such as nucleic acid extraction, real-time PCR, digital PCR and genome sequencing
A.1 Materials and equipment required:
● 50 or 100 mL centrifuge tubes
● Nuclease free micro-centrifuge tubes (1.5 mL)
● 200 µL, 20 µL and 10 µL filter micropipette tips
● Micropipettes
● Polyethylene glycol (PEG-8000), HIMEDIA
● Sodium Chloride (NaCl), Hi-AR% HIMEDIA
● Nuclease free water
● High speed refrigerated centrifuge
● Kimwipes/tissue paper
Vortex mixer
A.2 Protocol:
There are eight different environmental sample types, including poultry drinking water, wetland surface water, pond sediment, bird droppings, poultry litter, molted feathers, bird’s cage swab samples, and air, were systematically collected and processed for downstream analysis.
Note: Environmental samples such as poultry drinking water, wetland pond water, pond sediment and electrostatic dust clothes (EDC) are being processed for virus concentration method as mentioned below. The bird droppings, litter, molted feathers and air samples are being collected in virus transport media (VTM) are directly processed for viral RNA extraction(Table 1).
| Column | A | B | C | |
| Sr. No. | Sample types | Sampling method | PEG precipitation (Yes or No) | |
| 1 | Wetland water | Collect in 1 L Plastic Jerry can | Yes | |
| 2 | Wetland pond sediment | Collect in 50 mL falcons | Yes | |
| 3 | Poultry drinking water | Collect in 1 L Plastic Jerry can | Yes | |
| 4 | Electrostatic dust cloth (EDC) swab (from Poultry farm surface) | 5 cm x 5 cm blocks of sterile EDC used for poultry surface swab | Yes | |
| 5 | Poultry litter | Collect in 3 mL VTM | No | |
| 6 | Molted bird feathers | Collect in 3 mL VTM | No | |
| 7 | Bird droppings | Collect in 3 mL VTM | No | |
| 8 | Air | Collect using Sartorius MD8 Airport air sampler (sampling volume: 50 L/min for 20 Minutes= 1000 L) in Gelatin filters (final transfer in 6 mL VTM) | No |
Table 1: Details of sample types, sample collection methods and pre-processing methods used in wetland and poultry surveillance.
A.3 Sample pre-processing and viral enrichment Wetland water/ Poultrydrinking water: Take 200 mL of poultry drinking water or wetland surface water in sterile 200 mL polypropylene centrifuge tubes.Wetland pond sediment: Add 5 gm of sediment to 100 mL of autoclaved Milli-Q water in sterile centrifuge tubes. Bird’s cage swab: Take 50 mL of PBS in sterile centrifuge tubes in which swab was transferred (Squeeze out EDC using sterile spatula to recover maximum volume of PBS) (Note: Vortex all the samples thoroughly to ensure proper mixing)
Centrifuge the samples at 8000 rpm for 20 minutes to remove solid debris and particulate matter
Carefully transfer the clear supernatant to new sterile centrifuge tubes (100 mL or 200 mL, depending on the original sample volume), taking care not to disturb the pellet at the bottom
To the collected supernatant, add 10% (w/v) PEG 8000 and 5.58% NaCl.
Vigorously mix the contents by shaking or inverting the tube to ensure complete dissolution of PEG and NaCl.
Incubate the mixture at 4°C for 2 hours under static or shaking conditions in a refrigerator.
After incubation, centrifuge the tubes at 12,000 rpm for 90 minutes at 4°C.
Carefully decant the supernatant without disturbing the pellet at the bottom of the tube.
Gently remove any remaining liquid, taking care not to dislodge the pellet.
Re-suspend the pellet in 1 mL of nuclease-free water. Vortex briefly to ensure complete resuspension.
Store the re-suspended sample at -80°C until further use.
B Isolation of RNA from PEG:NaCl enriched pellets by Trizol + QIAamp Viral RNA mini kit
Materials and equipment required:
● DNAse, RNAse free micro centrifuge tubes (1.5 ml)
● Micropipettes with 200 µL, 20 µL and 10 µL filter micropipette tips
● Trizol reagent (Takara Bio, Cat.no 9108)
● Refrigerated centrifuge
● Ethanol (100%)
● Chloroform (100%)
● QIAamp Viral RNA MiniKit Qiagen, Cat no. / ID. 52904)
Protocol for RNA isolation using Trizol and QIAamp Viral RNA mini kit
Aliquot 200 µL of the sample from step 11 of A.3 and add 1 mL of Trizol reagent to it. Vortex the sample thoroughly. Incubate at room temperature for 5 minutes.
Add 200 µL of chloroform and Vortex briefly. Incubate at room temperature for 5 minutes. Centrifuge at 12000 X g for 15 minutes at 4℃.
Transfer the aqueous phase to a fresh RNAse-free micro-centrifuge tube.
Add equal volume of ethanol (96-100%) to the aqueous phase and mix by pulse-vortexing for 15 seconds. After mixing, briefly centrifuge the tube to remove droplets from the lid
Carefully transfer 600 μL of solution from step 4 to the QIAamp Mini column (in a 2 mL collection tube) without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Discard the tube containing the flow-through and place the QIAamp Mini column into a clean 2 mL collection tube.
Carefully open the QIAamp Mini column, and repeat step6. 5 for the remaining solution from step 6.4. Discard the flow through again and replace the column with a fresh 2 mL collection tube.
Carefully open the QIAamp Mini column, and add 500 μLLl of buffer AW1. Close the cap, and centrifuge at
6000 x g (8000 rpm) for 1 minute. Place the QIAamp Mini column in a clean 2 mL collection tube, and discard the tube containing the flow-through.
Carefully open the QIAamp Mini column, and add 500 μL of Buffer AW2. Close the cap and
centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 minutes.
To eliminate any chance of possible Buffer AW2 carryover, centrifuge at full speed (20,000 x g; 14,000
rpm) for 1 minute
Place the QIAamp Mini column in a clean 1.5 mL micro-centrifuge tube (not provided). Carefully open the QIAamp Mini column and add 40 μL of Buffer AVE (Elution buffer) equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min
Centrifuge at 6000 x g (8000 rpm) for 1 minute and store the elute at -20℃.
B. Isolation of total nucleic acid from poultry litter, bird droppings, molted feathers, and air samples
Materials and equipment required:
● MagMAX‱ Viral/Pathogen Nucleic Acid Isolation
Kit, Catalog no. A42352, Applied Biosystems by Thermo Fisher Scientific.
● 200 µL, 20 µL and 10 µL filter micropipette tips
● 1000 µL, 200 µL and 10 µL multichannel pipettes
● 1.5 mLl sterile eppendorf tubes
● KingFisher‱ Deepwell 96 well Plate (Catalog no.
97002820; Thermo Fisher Scientific)/ 1.5 or 2 ml sterile eppendorf tubes
● Kingfisher Flex System, Thermo Fisher Scientific
● Isopropanol
Components:
The following procedure uses components from theMagMAX‱ Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAX‱ Viral/Pathogen II Nucleic Acid Isolation Kit. Set up the instrument (200-μL sample input volume)
Ensure that the KingFisher‱ Flex Magnetic Particle Processor with 96 Deep-Well Head is set up with the KingFisher‱ Flex 96 Deep-Well Heating Block.
Ensure that the MVP_2Wash_200_Flex program has been downloaded from the product page and loaded onto the instrument.
Prepare the processing plates (200-μL sampleinput volume)
Prepare the processing plates according to the following table. Cover the plates with a temporary seal (such as MicroAmp‱ Clear Adhesive Film), then store at room temperature for up to 1 hour while you set up the sample plate.
Note: VTMs used for air sampling tend to solidify during storage at 4 ºC and must therefore be thawed at 37 ºC before proceeding with isolation.
| Column | A | B | C | D | E | |
| Sr. No. | Plate ID | Plate Position | Plate type | Reagent | Volume per well | |
| 1 | Wash 1 Plate | 2 | KingFisher‱ Deepwell 96 Plate | Wash buffer | 500 µL | |
| 2 | Wash 2 Plate | 3 | 80% Ethanol | 1000 µL | ||
| 3 | Elution Plate | 4 | KingFisher‱ 96 Plate | Elution solution | 50 µL | |
| 4 | Tip comb plate | 5 | Place a KingFisher‱ 96 tip comb for DW magnets in a KingFisher‱ 96 KF microplate |
Table 2: Details of components used in MagMAX‱ Viral/Pathogen Nucleic Acid Isolation.
Prepare Binding Bead Mix (200-μL sample input volume)
Prepare the required amount of Binding Bead Mix on each day of use.
1. Vortex the Total Nucleic Acid Magnetic Beads to ensure that the bead mixture is homogeneous.
2. For the number of required reactions, prepare the Binding Bead Mix according to the following table:
| Column | A | B | |
| Sr. No. | Components | Volume per well (µL) | |
| 1 | Binding Solution | 265 | |
| 2 | Total Nucleic Acid Magnetic Beads | 10 | |
| Total volume per well | 275 |
Table 3: Components of lysis solution used in MagMAX‱ Viral/Pathogen Nucleic Acid Isolation.
Mix well by inversion, then store at room temperature
Prepare a sample plate (200-μL sample input volume):
1. Add 5 µL of Proteinase K to each well in theKingFisher‱ Deepwell 96 Plate labeled "Sample Plate".
2. Add 200 µL of sample to each sample well.
3. Add 200 µL of Nuclease-free Water (not DEPC-Treated) to the Negative Control well.
4. Invert the Binding Bead Mix 5 times gently to mix, then add 275 µL to each sample well and the Negative Control well in the sample plate.
5. Finally add 200 μL of sample collected in VTM
Note: Remix Binding Bead Mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. The Binding Bead Mix is viscous, so pipet slowly to ensure that the correct amount is added. DO NOT reuse pipette tips to add Binding Bead Mix to the samples, as the high viscosity will cause volume variations.
Process the samples (200-μL sample input volume)
1. Select the MVP_2Wash_200_Flex on theKingFisher‱ Flex Magnetic Particle Processor with 96 Deep-Well Head.
2. Start the run, then load the prepared plates into position when prompted by the instrument.
3. After the run is complete (~22 minutes after start), immediately remove the Elution Plate from the instrument, and then cover the plate with MicroAmp‱ Clear Adhesive Film.
IMPORTANT! To prevent evaporation, seal the plate immediately.
C. Real-time reverse transcription-qPCR
C.1 Materials and equipment required:
● 200 µL, 20 µL and 10 µL filter micropipette tips
● 200 µL, 20 µL and 10 µL multichannel pipettes
● 8 well optical qPCR strips and caps
● 96 well optical qPCR plates
● Clear adhesive seals
● Primers and probes (Table 4)
● Nuclease free water
● QIAcuity One step advance MM (Qiagen, Cat no. / ID. 250131)
● Real time PCR system (7500Fast/7500/QuantStudio‱ 5 Real-Time PCR System, Thermo Fisher Scientific)
The extracted viral RNA will be further used for the detection of Influenza A Virus (IAV) using the following primers and probe.
| Column | A | B | |
| Sr. No. | Primer/Probe | Sequence/(-fluorophore) | |
| 1 | IAV-A F | 5’-CAAGACCAATCYTGTCACCTCTGAC-3’ | |
| 2 | IAV-A R | 5’-CATTTTGGATAAAGCGTCTACG-3’ | |
| 3 | IAV-A-P | FAM-5’-TGCAGTCCTCGCTCACTGGGCACG-BHQ1-3’ |
Table 4: Sequence information of Primer probe set used in qPCR-based IAV screening.
Setting up a qPCR run
1.Thaw qPCR reagents and samples on ice and briefly spin down before starting.
2. Set up a master mix for the number of samples to be tested plus a negative and positive control and one or two extra reactions to compensate for pipetting error.
| Column | A | B | C | |
| Sr. No. | Component | Volume (µl) | Final concentration | |
| 1 | QIAcuity one step advanced MM (4X) | 3 | 1X | |
| 2 | IAV-A FP from table (10 µM) | 0.3 | 0.25 μM | |
| 3 | IAV-A RP from table (10 µM) | 0.3 | 0.25 μM | |
| 4 | IAV-A probe from table (10 µM) | 0.15 | 0.125 μM | |
| 5 | RT enzyme mix (100X) | 0.12 | 1X | |
| 6 | Template/sample | 5 | - | |
| 7 | Nuclease free water | 3.13 | - | |
| Total volume | 12 |
Table 5: Components and their final volumes/concentrations used for setting up qPCR reactions
3.Aliquot 7 µLl of master-mix to 0.2 mlL 96-well qPCR plate or 0.2 ml 8-well optical strips.
4.Add 5 µLl of template to each of the wells to make up the final reaction volume of 12 µLl.
5.Seal the 96-well plate/ 8-well optical strips carefully with an optical qPCR plate adhesive seal/optical cap strip and then spin the plate/strips down briefly to gather all reagents at the bottom of the wells. Note: Make sure to remove any residual bubbles.
6.Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions:
| Column | A | B | C | |
| Sr. No. | No. of Cycle/s | Temperature °C | Duration | |
| 1 | 1 | 25 | 2 min | |
| 2 | 1 | 53 | 10 min | |
| 3 | 1 | 95 | 3 min | |
| 4 | 40 | 95 | 30 sec | |
| 5 | 60 | 30 sec (Data acquisition step) | ||
| 6 | Hold | 4 | ∞ |
Table 6: qPCR cycling conditions for IAV screening.
6. Once the run is completed, appropriately set the threshold above background noise, accordingly, assess the normalized Ct values for each gene target for each sample, determining positivity using the cut-off Ct values chosen from running the standards
E. Digital PCR assay
Materials and equipment required:
● 200 µL, 20 µL and 10 µL filter micropipette tips
● 200 µL, 20 µL and 10 µL multichannel pipettes
● 8-well PCR strips or 96 well PCR plates
● Primers and probes (Table 4)
● Nuclease-free water
● Digital PCR system (QIAcuity One QIAGEN, Cat no.
/ ID. 911001)
● QIAcuity Nanoplate 8.5 k ( Qiagen, Cat no. / ID. 250011) or 26-k (Qiagen, Cat no. / ID. 250001) 24 well plates
● Plate or microtube centrifuges
● Vortex
The extracted viral RNA will be further used for detection of IAV A using pre-optimized universal IAV-A primers and probe (Table 4) by digital PCR assay.
Setting up digital PCR run:
1.Thaw all digital PCR reagents on ice. Briefly vortex and spin down the reagents before starting the reaction setup.
2.Set up a master mix according to the number of samples to be tested, including appropriate negative and positive controls. (Note: Prepare the master mix for one or two extra reactions to compensate for pipetting errors).
| Column | A | B | C | |
| Sr. No. | Component | Volume (µLl) | Final concentration | |
| 1 | QIAcuity one step advanced MM (4X) | 3 | 1X | |
| 2 | IAV-A forward primer (10 µM) | 0.3 | 0.25 μM | |
| 3 | IAV-A reverse primer (10 µM) | 0.3 | 0.25 μM | |
| 4 | IAV-A probe (10 µM) | 0.15 | 0.125 μM | |
| 5 | RT enzyme mix (100X) | 0.12 | 1X | |
| 6 | Template RNA | 5 | - | |
| 7 | Nuclease free water | 3.13 | - | |
| Total volume | 12 | - |
Table 7: Digital PCR reaction mix components
Aliquot 7 µL of the prepared master-mix to 0.2 mLl 96-well PCR plates or 0.2 mLl PCR tubes
Add the appropriate templates, followed by negative and positive controls to the assigned wells containing the aliquoted master-mix. (Note: Care must be taken to avoid any cross contamination during template addition)
Seal the plate or tubes carefully with adhesive PCR plate seal/caps to conceal the prepared reaction mix. Briefly spin down the plates/strips to collect all reagents at the bottom of the well. (Note: Make sure to remove any bubblesformed during the reaction mix preparatory steps)
Gently transfer the prepared reaction mix from PCRplates/strips to respective nanoplate wells avoiding the generation of any bubbles. Seal the nanoplate wells appropriately with provided rubber adhesive seal. (Note: DO NOT centrifuge the nanoplate as it damages the chambers provided at bottom of nanoplate)
Set up the thermal cycling and final-step imaging parameters in the digital PCR system as provided in the table. Insert the sealed nanoplate inside the instrument and start the protocol.
| Column | A | B | C | |
| Sr. No. | Cycle | Temperature °C | Duration | |
| 1 | 1 | 50 | 40 min | |
| 2 | 1 | 95 | 3 min | |
| 3 | 40 | 95 | 5 sec | |
| 4 | 60 | 45 sec | ||
| Final imaging/data acquisition step |
Table 8: dPCR cycling conditions and imaging.
Data Collection and Analysis
1. After the run is completed, adjust the threshold line to obtain good separation between negative and positive partitions using auto thresholding.
2. When the positive and negative partitions are not well separated, automatic thresholding fails, and the threshold line must be manually adjusted. The threshold line can be adjusted manually by clicking into the different wells and adjusting the threshold line vertically. After adjusting the threshold, the analysis can be updated by selecting "recalculate"
3. Save the results of the run and Remove the plate from the instrument.
4. The obtained copy numbers/µL should be reviewed, and as per the dMIQE guideline, further final (total) copy numbersof targeted RNA should be calculated using the following formula: [(Total PCR
reaction volume in µL/volume of RNA template in µL used in PCR reaction) x (obtained copy numbers/µL)] here, (12 µL PCR reaction volume/5 µL RNA template) x (obtained copy numbers/µL) = x (obtained copy numbers/µL)
5. The obtained copy numbers of the targeted RNA must be taken into account for the interpretation of the results. If targeted RNA is detected in samples, it means that the samples contain targeted species, and if targeted RNA is not detected, it indicates that samples don't contain targeted amplicon
Protocol references
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2. Coombe M, Iwasawa S, Byers KA, Prystajecky N, Hsiao W, Patrick DM, Himsworth CG. A systematic review and narrative synthesis of the use of environmental samples for the surveillance of avian influenza viruses in wild waterbirds. The Journal of Wildlife Diseases. 2021 Jan 6;57(1):1-8. https://doi.org/10.7589/JWD-D-20-00082
3. Adlhoch C, Baldinelli F, Fusaro A, Terregino C. Avian influenza, a new threat to public health in Europe?. Clinical Microbiology and Infection. 2022 Feb 1;28(2):149-51. https://doi.org/10.1016/j.cmi.2021.11.005
4. Duan C, Li C, Ren R, Bai W, Zhou L. An overview of avian influenza surveillance strategies and modes. Science in One Health. 2023 Jan 1;2:100043. https://doi.org/10.1016/j.soh.2023.100043
Acknowledgements
This study was funded by the Gates Foundation . We also would like to thank the GEER(Gujarat Ecological Education and Research) Foundation, Department of Forest and Environment,Gandhinagar, for their support in wetland sampling.