Mar 30, 2026

Public workspaceSample Pre-processing, Nucleic acid extraction and screening (q/d-PCR) protocol for environmental surveillance of Lumpy skin disease virus (LSDV) in farm settings and bulked milk tank samples

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvrymmogmk/v1
  • Ramesh Pandit1,
  • Amrutalal Patel1,
  • Bhumika Prajapati1,
  • Tejas Shah1,
  • Kalpesh Katudia1,
  • Shewane Bishnoi1,
  • Abhinandan Patnaik1,
  • Jinal Thakore1,
  • Roselin Neihsial1,
  • Jill Gada1,
  • Rajvi Der1,
  • Parth Pandit1,
  • Kunjan Panchal1,
  • Bhagirath Dave1,
  • Harshal Purohit1
  • 1Gujarat Biotechnology Research Centre , Gandhinagar
Amrutalal Amrutlal Patel
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvrymmogmk/v1
Protocol Citation: Ramesh Pandit, Amrutalal Patel, Bhumika Prajapati, Tejas Shah, Kalpesh Katudia, Shewane Bishnoi, Abhinandan Patnaik, Jinal Thakore, Roselin Neihsial, Jill Gada, Rajvi Der, Parth Pandit, Kunjan Panchal, Bhagirath Dave, Harshal Purohit 2026. Sample Pre-processing, Nucleic acid extraction and screening (q/d-PCR) protocol for environmental surveillance of Lumpy skin disease virus (LSDV) in farm settings and bulked milk tank samples. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrymmogmk/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: January 27, 2026
Last Modified: March 30, 2026
Protocol Integer ID: 241610
Keywords: Lumpy Skin Disease Virus(LSDV), Environmental Surveillance , Gujarat Public Health, qPCR LSDV, lumpy skin disease virus, nucleic acid extraction, bulk milk sample, extracted dna, milk sample, milk tank sample, based extraction kit, viral particle, extraction kit, quantitative pcr, presence of lsdv, manger swab, lsdv, digital pcr, virus, pcr, time quantitative pcr
Funders Acknowledgements:
Gates Foundation
Grant ID: INV-064425
Abstract
Abstract: This protocol describes a standardized workflow for surveillance of Lumpy Skin Disease Virus (LSDV) from environmental and bulk milk samples. Multiple environmental samples, including drinking water, dung, slurry, leftover feed, and manger swabs, are processed using polyethylene glycol–NaCl precipitation to concentrate viral particles, followed by nucleic acid extraction using an optimized hybrid CTAB–silica column method. Milk samples are processed directly using a magnetic bead–based extraction kit. Extracted DNA is screened for the presence of LSDV using real-time quantitative PCR (qPCR) and digital PCR (dPCR).
Materials
Materials and equipments required For Processing
  1. 50 or 100 mL centrifuge tubes
  2. Chloroform treated microcentrifuge tubes (1.5 ml)
  3. 200 µL, 20 µL and 10 µL filter micropipette tips
  4. Micropipettes 1000 µL
  5. Polyethylene glycol (PEG-8000), molecular biology grade
  6. Sodium Chloride (NaCl), molecular biology grade
  7. Nuclease free water
  8. High speed refrigerated centrifuge
  9. Kimwipes/Tissue paper
  10. Vortex mixer
  11. Re-usable Kitchen Towel Roll, (Beco)

Materials and equipment required for nucleic acid isolation
  1. MagMAX‱ Viral/Pathogen Nucleic Acid Isolation Kit, CAT.NO. A42352, Applied Biosystems by Thermo Fisher Scientific.
  2. 200 µL, 20 µL and 10 µL filter micropipette tips
  3. 1000 µL, 200 µL and 10 µL Single and multichannel pipettes
  4. 1.5 mL sterile microcentrifuge tubes
Troubleshooting
(A) Sample processing and virus concentration
The aim of this step is to concentrate virus particles from environmental samples by using polyethylene glycol (PEG) and sodium chloride (NaCl). This process enhances the detection and further analysis of viruses by concentrating the sample for downstream applications such as nucleic acid extraction, realtime PCR, digital PCR, and genome sequencing.
Materials and equipments required:
50 or 100 mL centrifuge tubes DNAse-RNAse free microcentrifuge tubes (1.5 mL) 200 µL, 20 µL and 10 µL filter micropipette tips Micropipettes Poly(ethylene glycol) MW 8000-5KG HIMEDIA SKU: GRM7402
Sodium chloride (NaCl),Hi-AR‱ HIMEDIA GRM853-500G Nuclease free water High speed refrigerated centrifuge Kimwipes/Tissue paper
Vortex mixer Beco(Reusable Kitchen Towel Roll)


A.2 Protocol: There is total six types of environmental samples namely dung, leftover feed, slurry, drinking water, and manger swab (electrostatic dust cloth) collected and processed for further analysis. Note: Environmental samples such as drinking water, leftover feed, slurry, dung and manger swab are processed for virus concentration as mentioned below.
Different types of samples and their volume required for processing is mentioned in Table 1.

Table 1: Different types of samples, their volume required for processing, and methods concentration of virus particles

ABC
Sample types Sample amount PEG Precipitation Yes OR No
Drinking water 200 mL Yes
Leftover feed 5 gm in 100 mL NFW Yes
Slurry 5 ml in 95 mL NFW Yes
Dung 5 gm in 100 mL NFW Yes
Electrostatic dust cloth 5 cm blocks in 50 mL PBS Yes
Pooled milk/ Animal milk 200 µL No

Sample pre-processing

1Take 200 mL of drinking water in sterile200 mL polypropylene centrifuge tubes (e.g., centrifuge tubes).
2 Left over feed samples: Add 5mL of sample to 95 mL of autoclaved Milli-Q water in sterile falcons.
3 Manger swab: Take 50 mL of PBS in which EDC was transferred in sterile centrifuge tube
4 Vortex all samples thoroughly to mix properly.
5 Squeeze EDC using sterile spatula to recover maximum amount of PBS.
6 Centrifuge all samples at 8000 rpm for 20 minutes to remove solid debris.
7 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
Virus concentration
  1. To the collected supernatant, add 10% (w/v) PEG-8000 and 1 M NaCl.
  2. Vigorously mix the contents by shaking or inverting until PEG and NaCl dissolve completely.
  3. Carefully decant the supernatant without disturbing the pellet at the bottom of the tube.
  4. After incubation, centrifuge the tubes at 12,000 rpm for 90 minutes at 4°C.
  5. Carefully decant the supernatant without disturbing the pellet at the bottom of the tube.
  6. Gently remove any remaining liquid, taking care not to dislodge the pellet.
  7. Resuspend the pellet in 1 mL of nuclease-free water. Vortex briefly to ensure complete resuspension.
8 .Store the resuspended sample at -20°C until further use.

Isolation of total nucleic acid from milk samples (farm and bulk milk tank)
  1. Materials and equipments required:
  2. MagMAX‱ Viral/Pathogen Nucleic Acid Isolation Kit, CAT.NO. A42352, Applied Biosystems by Thermo Fisher Scientific.
  3. 200 µL, 20 µL and 10 µL filter micropipette tips
  4. 1000 µL, 200 µL and 10 µL Single and multichannel pipettes
  5. 1.5 mL sterile microcentrifuge tubes


Components:

The following procedureuses components from the MagMAX‱Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAX‱ Viral/Pathogen II NucleicAcid Isolation Kit.
Set up the instrument (200-μL sample input volume)
  1. 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.
2 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) sample input 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.
ABCDE
Plate ID Plate Position Plate type Reagent Volume per well
Wash 1 Plate 2 KingFisher™ Deepwell 96 Plate Wash Buffer 500 µL
Wash 2 Plate 3 80% Ethanol 1 mL
Elution Plate 4 KingFisher™ 96 well Plate Elution solution 35 µL
Tip comb plate 5 Place a KingFisher‱ 96 tip comb for DW magnets in a KingFisher‱ 96 KF microplate
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:
Component Volume per sample (µL)
Binding Solution 265
Total Nucleic Acid Magnetic Beads 10
Total volume per well 275
Mix well by inversion, then store at room temperature.

Prepare sample plate (200-μL sample input volume) Add 5 µL of Proteinase K to each well in the KingFisher‱ Deepwell 96 Plate labeled "Sample Plate". Add 200 µL of sample to each sample well. Add 200 µL of Nuclease-free Water (not DEPC-Treated) to the Negative Control well. Invert the Binding Bead Mix 5times gently to mix, then add 275 µL to each sample well and the Negative Control well in the sample plate.

Note: Remix Binding Bead Mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. 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 thehigh viscosity will cause variations in the volumes added.

Process the samples 200-μLsample input volume

  1. Select the MVP_2Wash_200_Flex on the KingFisher‱ 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 (~22minutes after start), immediately remove the Elution Plate from the instrument, and then cover the plate with MicroAmp‱ ClearAdhesive Film.
IMPORTANT! To prevent evaporation, seal the elution immediately.

Isolation of DNA from environmental samples (dung,slurry, drinking water, manger swab and left over feed by Hybrid DNA Extraction Protocol combining CTAB Lysis and QIAamp Fast DNA Stool Mini Kit

CTAB-kit method:

Materials
  1. Resuspended enriched PEG precipitate 200 µL
  2. Centrifuge
  3. Dry bath/water bath (70°C and 56.5 °C)
  4. QIAamp Fast DNA Stool Mini Kit by Qiagen Cat. No ID.51604
  5. 5M NaCl (Mol Bio Grade)
  6. 10% SDS( Mol Bio Grade)
  7. CTAB
  8. Phenol
  9. Chloroform
  10. Iso amylalcohol
  11. NFW

Procedure

  1. Aliquot sample Pipette 200 µL of environmental sample lysate into a microcentrifuge tube and add 20ul proteinase K and incubate at 56 °C for 30 minutes.
  2. CTAB extraction Perform the standard CTAB protocol as previously established. The steps are mentioned below.
Prepare cell lysis buffer as below
a) Add 50 µL of 10% SDS
b) Add100 µL of 5 M NaCl
c) Add 160 µL CTAB
d) Mix and vortex well

Phenol–chloroform extraction
a) Add 520 µL PCI (Phenol: Chloroform: Isoamyl alcohol)
b) Vortex thoroughly
c) Centrifuge at 12,000 rpm for 10 minutes
d) Carefully transfer the aqueous phase to a new tube

3 Remove inhibitors
  1. Add 200 µL of InhibitEX to the supernatant.
  2. Vortex thoroughly for 1 minute.
3. Incubate the mixture at 70 °C for 10 minutes.
4.Clarify lysate
6 Carefully transfer the clear supernatant to a new microcentrifuge tube.
6 Carefully transfer the clear supernatant to a new microcentrifuge tube.
7 Add the supernatant to the column provided with the QiAMPViral DNA stoolkit and follow the instructions provided by the manufacturer.
8 Elute the DNA in 35uL NFW

qPCR-Applied Biosystems 7500 Real-Time PCR System and Applied Biosystems 7500 fast real time PCR system
Materials and equipments required:

200 µL, 20 µL and 10 µL filter micropipette tips
200 µL, 20 µL and 10 µL singlechannel and multichannel pipettes
96 well PCR plates
Clear adhesive seals
Primers and probes
Nuclease free water
200 µL, 20 µL and 10 µL single channel and multichannel pipettes


The extracted DNA will be screened for detection of Lumpy Skin Disease Virus (LSDV)using the following primers and probe Table 1:
AB
Primer/Probe Sequence/(-flourophore)
LSDV-059-F 5’- TGAATTAGTGTTGTTTCTTC-3’
LSDV-059-R 5’-GGGAATCCTCAAGATAGTTCG-3’
LSDV-59-P FAM 5’-TGCCGCAAAATGTCGA-3’ MGB
LSDV-091 F* 5’-CAACCAACAATAACTATTATCTGAA-3’
LSDV-091 R* 5’-TTTCTTCTCCTAAATCAGTTGC-3’
LSDV-091 SP* ROX 5’-TCCGCCACAGATATGT-3’ MGB
LSDV-044 F* 5’- AAACAATCGTAACTAATCCA-3’
LSDV-044 R*5’-TGGAGTTTTTATGTCATCGTC-3’
LSDV-044 P*FAM 5’-TCGTCGTCGTTTAAAACTGA-3’ QSY
*Currently we are using the primer LSDV-059-F, R and its probe, however other primers can also be used.
Setting up qPCR run


1Thaw 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 controls and one or two extra reactions to subtract for pipetting error.


Component Volume (µL) Final concentration
Probe PCR master mix (4X) 3 1X
Forward primer from Table 1 (10 µM) 0.3 0.25 μM
Reverse primer from Table 1 (10 µM) 0.3 0.25 μM
Probe from Table 1 (10 µM) 0.125 0.125 μM
Template/sample 5 -
Nuclease free water 3.275 -
Total volume 12 -
3 Aliquot 20µL of mastermix to each required well in a 96-well plate or 8-welloptical strips and caps.
4 Seal the plate carefully with an optical plate adhesive seal the spin the plate down briefly to gather all reagents at the bottom of the well and remove bubbles.
5 Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions



PCR Cycling conditions

Cycle Temperature °C Duration
1 95 5 minutes
40 95 30 seconds
55 30 seconds
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.
Digital PCR QIAcuity One QIAGEN Cat no. / ID. 911001
Materials and equipments required:
  1. 200 µL, 20 µL and 10 µL filter micropipette tips
  2. 200 µL, 20 µL and 10 µL multichannel pipettes
  3. PCR tubes or 96 well plates
  4. Primers and probes(Eurofins)
  5. Nuclease free water
  6. Digital PCR system
  7. 8.5k or 26-k partitions nanowell plates
  8. Plate or microtube centrifuges
  9. Vortex

DO NOT centrifuge the nano well plate as it damages the chambers provided at bottom of nanowell plate
Following primers and probe are used in digital PCR assay.

Primer/Probe Sequence/(-flourophore)
LSDV-059-F 5’- TGAATTAGTGTTGTTTCTTC-3’
LSDV-059-R 5’-GGGAATCCTCAAGATAGTTCG-3’
LSDV-59-P FAM 5’-TGCCGCAAAATGTCGA-3’ MGB
LSDV-091 F* 5’-CAACCAACAATAACTATTATCTGAA-3’
LSDV-091 R* 5’-TTTCTTCTCCTAAATCAGTTGC-3’
LSDV-091 SP* ROX 5’-TCCGCCACAGATATGT-3’ MGB
LSDV-044 F* 5’- AAACAATCGTAACTAATCCA-3’
LSDV-044 R* 5’-TGGAGTTTTTATGTCATCGTC-3’
LSDV-044 P* FAM 5’-TCGTCGTCGTTTAAAACTGA-3’ QSY
*Currently we are using the primer LSDV-059-F, R and its probe, however other primers can also be used.

Setting up digital PCRrun:

  1. Set up a master mix for the number of samples to be tested plus a negative and positive controls and one or two extra reactions to subtract for pipetting error.
  2. Set up a master mix for the number of samples to be tested plus a negative and positive controls and one or two extra reactions to subtract for pipetting error.


Component Volume (µL) Final concentration
QIAcuity Advance probe MM (4X) 3 1X
Forward primer from Table 1 (10 µM) 0.3 0.25 μM
Reverse primer from Table 1(10 µM) 0.3 0.25 μM
Probe from Table 1 (10 µM) 0.15 0.125 μM
RT enzyme mix (100X) 0.12 1X
Template DNA 5 -
Nuclease free water 3.13 -
Total volume 12 -
3 Aliquot 7 µL of master mix to each required well in a 96-well plate or 0.2 mL PCR tubes.
4 Aliquot 7 µL of master mix to each required well in a 96-well plate or 0.2 mL PCR tubes/wells to minimize the contamination.
5Seal the plate or tubes carefully with adhesive seal the spin the plate down briefly to gather all reagents at the bottom of the well and remove bubbles.
6Carefully transfer the mastermix containing samples to respective nano well plates to avoid the bubbles generation and seal the nano well plate appropriately with provided rubber adhesive seal.

DO NOT centrifuge the nano well plate as it damages the chambers provided at bottom of nanowell plate.

7 Start the digital PCR instrument after setting it up appropriately and carry out cycling using the following conditions to instrument software. Insert the sealed nano well plate inside the instrument and start the protocol as provided.

PCR Cycling conditions
ABC
Cycle Temperature °C Duration
1 95 5 minutes
40 95 30 seconds
60 30 seconds
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 Ct less than 35 is considered positive /suspected and Ct between35-40 is considered as suspected. 5 The obtained copy numbers/µL should be reviewed, further final (total) copy numbers of targeted DNA should be calculated using the following formula: [(Total PCR reaction volumein µL/volume of DNA template in µL used in PCR reaction) x (obtained copy numbers/µL)] here, (12 µL PCR reaction volume/5 µL DNA template) x (obtained copy numbers/µL) = x (obtained copy numbers/µL).

Protocol references
Acknowledgements
Gates Foundation and Department of Animal Husbandry , Government of Gujarat