Nov 09, 2024
PCR-based cytotyping to detect Wolbachia in insect tissues
- Brandon S. Cooper1,
- J. Dylan Shropshire2
- 1Division of Biological Sciences, University of Montana;
- 2Department of Biological Sciences, Lehigh University
- Shropshire Lab
Protocol Citation: Brandon S. Cooper, J. Dylan Shropshire 2024. PCR-based cytotyping to detect Wolbachia in insect tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49yx8go5/v1
Manuscript citation:
Shropshire JD, Conner WR, Vanderpool D, Hoffmann AA, Turelli M, Cooper BS. (2024) Rapid host switching of Wolbachia and even more rapid turnover of their phages and incompatibility-causing loci. bioRxiv. 12.04.569981. PMCID: PMC10723362
Shropshire JD, Hamant E, Conner WR, Cooper BS. (2022) cifB-transcript levels largely explain cytoplasmic incompatibility variation across divergent Wolbachia. PNAS Nexus. 1(3):pgac099. PMCID: PMC9364212
Hague MTJ, Shropshire JD, Caldwell CN, Statz JP, Stanek KA, Conner WR, Cooper BS. (2022) Temperature effects on cellular host-microbe interactions explain continent-wide endosymbiont prevalence. Curr Biol. 32(4):878. PMCID: PMC8891084
Shropshire JD, Hamant E, Cooper BS. (2021) Male age and Wolbachia dynamics: investigating how fast and why bacterial densities and cytoplasmic incompatibility strengths vary. mBio. 12(6):e02998-21. PMCID: PMC8686834
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: September 11, 2024
Last Modified: November 09, 2024
Protocol Integer ID: 107441
Keywords: Drosophila, Wolbachia, cytotyping, infection check, PCR
Abstract
This protocol details a PCR-based method for detecting Wolbachia in insect tissues. By amplifying the wsp gene, specific to Wolbachia, and the host 28s rDNA gene as a control, this technique allows for accurate identification of Wolbachia-bearing and Wolbachia-free samples. Proper interpretation of control samples is crucial to ensure reliable results.
Guidelines
Unless otherwise specified, steps are performed at room temperature in a 1.5 mL PCR-clean Eppendorf tube containing the appropriate tissue. Precautions are taken throughout to protect samples against contamination. All centrifuge steps are at 12,000 x g.
Materials
Materials
- Agarose, electrophoresis grade (Fisher Scientific, BP-160)
- Centrifuge tubes, safe-lock, PCR clean, 1.5 mL (Eppendorf, 022363212)
- Ceramic beads, 2.8mm (VWR, 19-646-3)
- DNA ladder (Thermo Fisher Scientific, SM1552)
- EDTA (PR1MA, KCE57020-250)
- Erlenmeyer flask, 250 mL (any)
- Ethidium bromide (Thermo Fisher Scientific, 15585011)
- GoTaq Green (Promega, M7123)
- Graduated cylinder (any)
- LB Buffer, 20x (Faster Better Media LLC, LB20-4)
- NaCl (Apex BioResearch, DB0483)
- Pipette tips, 0.1-10 μL (Eppendorf, 0030078500)
- Pipette tips, 2-100 μL (Eppendorf, 0030078543)
- Pipette tips, 50-1000 μL (Eppendorf, 0030078578)
- PCR tubes/plates and caps/seals (any)
- Primers (wsp & 28s)
- Proteinase K (Thermo Fisher Scientific, 25530049, ThermoFisher)
- Screw-top jar, Glass, 500 mL (any)
- Tris-HCl (1M, pH = 8.0) (Thermo Fisher Scientific, J22638-AE)
- Water, distilled (PR1MA, KCW20525-10000)
- Water, molecular biology grade (MidSci, 786-73C)
Equipment
- Analytical balance (any)
- Autoclave
- Bead-mill homogenizer (Benchmark, IPD9600)
- Centrifuge (Eppendorf, 5420)
- Electrophoresis power supply (Thermo Fisher Scientific, PS0600)
- Electrophoresis system (Thermo Fisher Scientific, OWL)
- Freezer (any)
- Fridge (any)
- Gel imager (any)
- Microwave (any)
- Pipette, 10 μL (Eppendorf, 4861000708)
- Pipette, 100 μL (Eppendorf, 4861000716)
- Pipette, 1000 μL (Eppendorf, 4861000732)
- Thermocycler (Bio-Rad, C1000)
- Thermomixer (Eppendorf, F2.0)
Safety warnings
This protocol involves the use of various chemicals and reagents that require careful handling and strict adherence to safety guidelines to ensure safe laboratory practices. Please review the Material Safety Data Sheets (MSDS) for each reagent before beginning the protocol and take appropriate precautions. All steps should be performed while wearing gloves, a lab coat, and eye protection.
Before start
Sterilize the working surface with 10% bleach and 70% ethanol.
Prepare squish buffer
Prepare squish buffer
Mix the following in a 500 mL glass screw-top jar. Use a graduated cylinder to measure liquids and an analytical balance to measure solids.
- 20 mL Tris-HCl (1 Molarity (M) , pH = 8.0)
- 0.0744 g EDTA
- 0.2922 g NaCl
- 180 mL diH2O
Autoclave 00:30:00 on fluid cycle to sterilize. Make sure the screw top is loose. Allow to cool to room temperature.
30m
Add 300 µL of Proteinase K. Mix by gently shaking the bottle.
Create 15 mL aliquots labeled with the date.
Store at 4oC until use.
Sample collection, homogenization, and lysis
Sample collection, homogenization, and lysis
Collect samples by adding three ceramic beads (2.8 mm) and flies to a 1.5 mL centrifuge tube
Homogenize flies for 00:02:00 at 1500 rpm using a bead-mill homogenizer.
2m
Add 50 µL of squish buffer per Drosophila fruit fly.
Incubate at 65 °C for 00:45:00 at 300 rpm to lyse cells using a ThermoMixer.
45m
Incubate at 94 °C for 00:04:00 at 300 rpm to inactivate Proteinase K.
4m
Debris precipitation, storage, and use
Debris precipitation, storage, and use
Centrifuge for 00:02:00 to pellet cellular debris.
2m
Store squish-extracted DNA at -20 °C for 2 weeks or at 4 °C for 24 hours.
Note
Considerations for use:
- Centrifuge the DNA before use to bring any protein/fly pieces to the bottom of the tube.
- When pipetting, take care to only collect the liquid on the top of the mixture.
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR)
Create two PCR master mixes with a final reaction volume of 10 µL with 2 µL of DNA template. Each sample should be tested with wsp primers (for Wolbachia) and 28s (for the host).
- wsp_F: 5'-TGGTCCAATAAGTGATGAAGAAAC-3'
- wsp_R: 5'-AAAAATTAAACGCTACTCCA-3'
- 28s_F: 5'-TACCGTGAGGGAAAGTTGAAA-3'
- 28s_R: 5'-AGACTCCTTGGTCCGTGTTT-3'
Note
Plan for the inclusion of a positive control known to have Wolbachia, a negative control without Wolbachia, and a no template control where water is added in place of a DNA template.
Add 8 µL of the master mix to each well.
Add 2 µL of the DNA template to each well.
Cap or seal wells. Centrifuge for 00:00:10 at 12000 rcf . Transfer to a thermocycler.
10s
Perform PCR following the relevant program.
- wsp program: 94°C for 5 min, (94°C for 30 sec, 55°C for 30 sec, and 72°C for 75 sec)x40, 72°C for 5 min, hold at 4°C
- 28s program: 94°C for 2 min, (94°C for 30 sec, 50°C for 30 sec, and 72°C for 60 sec)x40, 72°C for 8 min, hold at 4°C
Make 1% LB Gel
Make 1% LB Gel
Seal gel tray in preparation of adding liquid agarose.
Add 100 mL 1x LB Buffer to a 250 mL Erlenmeyer flask. Add 1 g agarose to the flask. Swirl to mix.
Microwave until just boiling. Stop the microwave. Swirl the flask to mix. Look for flakes of unmelted agarose. Continue until fully dissolved.
Note
Caution: The glass will be hot, and the agarose can boil over if not observant.
Run tap water over the flask for 00:00:30 to cool. Swirl the flask while cooling.
30s
Add 2 drops of ethidium bromide to the flask and mix well by swirling. Pour liquid agarose gel into the gel tray and add comb(s). Be careful not to create bubbles in the gel with the comb.
Wait 00:20:00 for the gel to solidify
20m
Carefully remove the combs and unseal the gel tray.
Perform gel electrophoresis with PCR products
Perform gel electrophoresis with PCR products
Load gel and tray into gel box. Add 1x LB Buffer until the fill line.
Add 3 µL of the ladder into the furthest left well on each row.
Add 3 µL of each PCR sample into each well.
Note
- Work quickly or the samples will start to disperse in the wells.
- Be careful not to poke the pipet tip into the side or bottom of the well.
Cover the gel box and attach it to a power supply. Run the gel at 300 Volts. Monitor the migration of the loading dyes to determine when to stop.
Image the gel.
Interpret the control samples. If the controls are as expected, proceed to interpret the experimental samples.
Interpreting no-template control (NTC):
- Expected result: No bands for wsp or 28s.
- Unexpected result: Any band indicates reagent contamination. Cytotyping inconclusive.
Interpreting Wolbachia-free DNA (negative control):
- Expected result: No wsp band, clear 28s band.
- Unexpected result: wsp band indicates sample contamination during collection or processing. No 28s band indicates poor DNA quality, PCR issues, or errors in sample loading. Cytotyping inconclusive.
Interpreting Wolbachia-containing DNA (positive control):
- Expected result: Clear bands for both wsp and 28s.
- Unexpected result: No bands indicate poor DNA quality, PCR issues, or errors in sample loading. Cytotyping inconclusive.
Interpreting test samples:
- + wsp, + 28s: Wolbachia-positive
- - wsp, + 28s: Wolbachia-negative
- + wsp, - 28s: Potentially Wolbachia-positive, but technical or human errors may have affected the 28s reaction.
- - wsp, - 28s: Inconclusive. Technical or human errors likely impacted both reactions.
Protocol references
Oligos reported in this protocol originate from Braig et al. 1998 and Nice et al. 2009.
Braig HR, Zhou WG, Dobson SL, O’Neill SL. (1998) Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol. 180(9):2373–2378. PMID: 9573188
Nice CC, Gompert Z, Forister ML, Fordyce JA. (2009) An unseen foe in arthropod conservation efforts: The case of Wolbachia infections in the Karner blue butterfly. Biol Conserv. 142(12):3137–3146.