Jul 18, 2025

Public workspaceA standardised protocol for extracting environmental DNA (eDNA) from honey samples of diverse origin

DOI
dx.doi.org/10.17504/protocols.io.14egnr816l5d/v1
  • Gopika Kottantharayil1,
  • Travis Beddoe2,
  • Dr. Gemma Zerna2
  • 1Student;
  • 2Head of Department- Ecological Plant and Animal Sciences
  • Dr. Gemma Zerna: Associate Lecturer - Ecological Plant and Animal Sciences;
Gopika Kottantharayil
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DOI: https://dx.doi.org/10.17504/protocols.io.14egnr816l5d/v1
Protocol Citation: Gopika Kottantharayil, Travis Beddoe, Dr. Gemma Zerna 2025. A standardised protocol for extracting environmental DNA (eDNA) from honey samples of diverse origin. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnr816l5d/v1
Manuscript citation:
  1. Bhasi, G., Zerna, G., & Beddoe, T. (2025). Nationwide Screening for Arthropod, Fungal, and Bacterial Honey Bee Pathogens: Utilizing Environmental DNA from Honey samples in Australia. DOI:10.20944/preprints202505.1437.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: July 18, 2025
Last Modified: July 18, 2025
Protocol Integer ID: 222751
Keywords: extracting environmental dna, backyard hive honey sample, environmental dna, hive honey sample, honey samples of diverse origin, honey sample, edna isolation, commercial honey, edna, standardised protocol, house protocol
Abstract
We have optimised and validated an in-house protocol for extracting environmental (eDNA) that is applicable to both processed (Commercial honey samples) and unprocessed (backyard hive honey samples) honey samples. The protocol comprises a pre-treatment phase and post-treatment phase for eDNA isolation.
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Image Attribution

Flow diagram showing the pre-treatment and post-treatment phase in eDNA extraction from honey samples.



Guidelines
1. Honey needs to be completely homogenized.
2. Vortex well to make sure no clumps of honey are left in the tubes.
3. Complete the whole extraction process in a single day.
4. The samples can be weighed (12g of the same honey sample in 4 different 50 mL tubes), which can be done the day before extraction.
5. The TNE extraction buffer should be pre-heated before adding it to the pellet. Do not heat for more than 5 minutes.



Materials
  1. Honey samples
  2. Ultra-pure water
  3. 50 mL tubes
  4. 5 mL tubes
  5. 1.5 mL and 2 mL tubes
  6. 5 mm Glass beads
  7. TNE Buffer (pH 7.5)
  8. 5 M guanidine hydrochloride (w/v)
  9. Proteinase K solution (20 mg/ml-1)
  10. 6M sodium iodide (NaI)
  11. 100 mg/mL silica dioxide (SiO2)
  12. Silica wash buffer
  13. 10 mM Tris-HCl Elution Buffer (pH 8)
Troubleshooting
Safety warnings
1. Do not leave the pellet with elution buffer in water bath for more than 5 minutes.
Before start
1. Make sure the TNE buffer is made freshly and stored at 4°C.
2. Proteinase K has to be kept on ice during the process.

Pre-Treatment Phase
A total of 50 g of honey was carefully divided into four sterile 50 mL centrifugation tubes, with 12.5 g transferred into each tube.Amount50 g

40 mL of ultrapure water (Milli-Q) is added to each tube and thoroughly vortexed to ensure complete homogenization.Amount40 mL

Samples are incubated in a water bath at 40°C for 10 minutes.Temperature40 °C

Samples are then centrifuged at 4700 ×g for 35 minutes.Centrifigation4700 x g , 35 minutes

Following centrifugation, the supernatant is carefully discarded, and each resulting pellet is resuspended in 5 mL of ultrapure water.Amount5 mL

The suspensions were pooled into sterile 50 mL centrifugation tubes corresponding to the original honey samples.Amount50 mL

The pooled samples are then subjected to an additional centrifugation at 4700 ×g for 30 minutes.Centrifigation4700 x g , 30 minutes

After centrifugation, the supernatant is carefully discarded, and the pellet is resuspended in 500 µL of ultrapure water.Amount500 µL

This suspension is then transferred to a 2 mL microcentrifuge tube containing seven 5 mm sterile glass beads.
The microcentrifuge tubes are vortexed for 2 minutes.
The glass beads are removed using sterile, fine-tipped tweezers.
The suspension was then centrifuged at 11,000 × g at 4 °C for 10 minutes.Centrifigation11000 x g, 4°C , 10 minutes

The supernatant is carefully discarded, and the resulting pellet serves as the starting material for the post-treatment phase.

Post-Treatment Phase
The pellet obtained from the pre-treatment phase is resuspended in 860 µL of pre-heated (60 °C) TNE extraction buffer (10 mM Tris-HCl, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 1% (w/v) sodium dodecyl sulfate, pH 7.5).Amount860 µL

To the lysate, 100 µL of 5 M guanidine hydrochloride (w/v) and 40 µL of proteinase K solution (20 mg/mL-1) were added to facilitate protein denaturation and prevent the enzymatic degradation of DNA.Amount100 µL
Amount40 µL

The lysis mixture is incubated at 60 °C for 3 hours in a Thermomixer Comfort (Eppendorf AG, Hamburg, Germany) with agitation at 900 RPM.Shaker900 rpm, 60°C 3 hour

Followed by centrifugation for 15 minutes at 17,000×g at 4° C.Centrifigation17000 x g, 4°C , 15 minutes

The supernatant is collected into a 5 mL tube for DNA purification.
The supernatant is mixed with 2 Volumes of 6 M sodium iodide (NaI) and 100 µL of 100 mg/mL silica dioxide (SiO2).Amount2 V
Amount100 µL

The samples are allowed to mix well by gentle agitation on a rocker for 30 minutes.
This is followed by centrifugation for 10 minutes at 5000 ×g at 4° C.Centrifigation5000 x g, 4°C , 10 minutes

The supernatant is carefully discarded.
The pellet is resuspended in 500 µL of silica wash buffer (50% (v/v) ethanol, 10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 8) by vortexing.Amount500 µL

This suspension is then centrifuged for 1 minute at 4700 ×g at 4° C.Centrifigation4700 x g, 4°C , 1 minute

The wash step (step 23 and 24) is repeated two more times.
The supernatant is carefully discarded.
To elute eDNA from the silica matrix, 50 µL of elution buffer (10 mM Tris-HCl,  pH 8) is added and incubated at 70°C for 5 minutes. Amount50 µL

Following the incubation, the samples are centrifuged for 5 minutes at 16,000 ×g.Centrifigation16000 x g , 5 minute

The supernatant is carefully pipetted into a 1.5 mL microcentrifuge tube and stored in -20 ° C.Temperature-20 °C

The concentration of isolated eDNA is measured using a Nanodrop Eight spectrophotometer.
The isolated eDNA is validated by performing end-point Polymerase Chain Reaction (PCR) targeting Apis mellifera mtDNA. Genomic DNA from a healthy adult bee is used as a positive control, and nuclease-free water as a no-template control (ntc).
Amplified PCR products are visualized by agarose gel electrophoresis using a 2% (w/v) agarose gel prepared in  1X TBE buffer (0.13M Tris, 45 Mm Boric acid, 2.5 mM EDTA pH 7.6 ) containing 0.2 µg/mL ethidium bromide.
The DNA bands are visualized under a UV illuminator to confirm successful amplification.
Protocol references
1. Soares,S., Amaral, J. S., Oliveira, M. B. P. P., & Mafra, I. (2015, 2015/2//). Improving DNA isolation from honey for the botanical origin identification. Food control, 48, 130-136. https://doi.org/10.1016/j.foodcont.2014.02.035
2. Mafra,I., Silva, S. A., Moreira, E. J., da Silva, C. S. F., Beatriz, M., & Oliveira, P. (2008). Comparative study of DNA extraction methods for soybean derived food products. Food control, 19(12), 1183-1190.
Acknowledgements
This study is supported by the La Trobe University-Shah Rukh Khan scholarship, research support from my supervisors, Prof. Travis Beddoe and Dr. Gemma Zerna.