Expertise needed to implement the protocol: This protocol is appropriate for anyone with familiarity with routine real-time PCR reactions.
Limitations: PCR amplification of retrotransposons has similar sensitivity to many methods currently used in modern forensics. The key to the successful utilization of this method is contamination prevention, more so than just ensuring that there is no carryover of material between samples during extraction or of nucleic acids during PCR setup. Contamination may arise at any step in the process from the handling of the ticks during collection and sorting. Throughout this protocol, measures used to prevent contamination will be detailed. These may seem “over-the-top” for many veteran researchers with years of experience setting up successful PCRs. However, these measures were arrived at because problems arose in our laboratory. Since these strict measures have been implemented, contamination has ceased to be an issue.
This method works best if the ticks are young and fresh. We have found that the success of this method decreases with the age of the tick10. Do not allow collected ticks to sit alive in incubators in the lab waiting to be processed. Freeze the ticks soon after collection. Because ticks are promptly killed, there is no need to put plaster or anything else in the bottom of collection tubes (which is usually done to help modulate humidity in the tubes to keep the ticks alive). We use 3-dram snap cap vials (VWR 89097-516) for tick collections. This facilitates the ability to externally decontaminate the ticks directly in the collection tubes (see below). Depending on local circumstances, environmental DNA (eDNA) may contaminate the exterior of questing ticks. It is also likely that questing ticks may be contaminated by DNA from phoretic hosts 13.
I. Tick collection
Contamination prevention procedures for tick collection: During our trips to the field, we often trap animals and sample mouse boxes in addition to collecting ticks. It is important that all equipment used for activities involving animals are kept separate from the tick-collecting equipment. Bloodmeal identification PCRs may become positive simply from the use of contaminated forceps, such as ones that were also used to remove ticks from animals. Use clean forceps that are dedicated to tick collections and are not used for any other task. We routinely store our tick drags inside plastic trash bags; to keep them protected from the other equipment (such as mouse traps) that is also stored in the back of our field vehicle. Collection tubes for ticks should also be stored separately from the tubes and other supplies used to work-up field-collected animals. Tick drags should periodically be placed in direct sun for several hours to inactivate environmental DNA.
II. Tick sorting and nucleic acid extraction.
Contamination prevention procedures for processing ticks in the lab: The processing of the ticks must be done in an area physically separated from the PCR machines. This area must also be free of all possible mammalian contamination. Activities such as working next to someone extracting mouse tissues or working in a hood that is also used for mammalian tissue-cultures must be avoided. Be aware of contaminating aerosols from centrifuges or other common equipment that have exhaust or cooling fans. The use of extraction robots may be another source of contamination, if the robot is used for diverse samples (e.g., to extract mouse blood, cell cultures, ticks, mosquitoes). All forceps, racks, pipettors, lab benches etc…should be dedicated to tick work, or at the minimum be decontaminated before use (use bleach or DNA Away (Fisher #21-236-28)). Finally, consider your clothes. Be mindful that fur and dander from your pets are all over your clothes and can cause contamination of the samples. Be sure to, at the very least, wear a clean lab coat that is frequently changed.
Freshly diluted 5% bleach
Molecular biology grade water (UltraPure DNase/RNase free distilled water Invitrogen #10977015)
Snap-cap microcentrifuge tubes (Eppendorf #022364111)
100 micron zirconium beads (Ops Diagnostics #BLBZ 10025014)
1000µl pipette tips tips- heat sealed (Fisher Scientific #02-707-403)
Aerosol barrier pipette tips
Transfer pipettes (Fisher Scientific #13-711-7M)
Alcohol burner (Fisher #04-245-1)
QuickExtract (Lucigen Corp.#QE09050)
1. Don clean gloves and a clean lab coat.
2. Heat seal the tip of 1000µl pipette tips by holding them briefly in the flame of an alcohol burner. (Do not blow on the tip to extinguish the flame, wave it!) Reinsert the sealed tip into the tip box and repeat until the entire box is done.
3. To minimize freezing and thawing of ticks, only remove from the freezer the number of ticks that can be successfully sorted and extracted at once.
4. Decontaminate the outer surface of the ticks by soaking in 5% bleach for 2 minutes.This can be done directly in the collection vial. Fill the vial ¾ full with 5% bleach. Ticks may float; mix by inverting the tube so that the bleach reaches all sides.
5. Decant the bleach using a transfer pipette.
6. Wash for 30 seconds in PCR-grade water. Fill the collection vial ¾ with water. Invert to mix.
7. Decant the water using a clean transfer pipette.
8. Repeat water wash 3 more times.
9. Sort ticks into individual 1.5ml Eppendorf tubes using clean dedicated forceps.
10. Homogenize each tick individually using a heat-sealed 1000µl tip in 2µl water with 100 micron zirconium beads to facilitate grinding.
11. Extract tick homogenate for DNA/RNA.
Nucleic acid extraction can be done using any published procedure. Bloodmeal host DNA has been successfully extracted from ticks using HOTSHOT14 and DNEasy kits (Qiagen, Inc.). Our current method uses QuickExtract (Lucigen Corp.) and is a quick and easy method that will yield both DNA as well as RNA. This method is detailed below.
Protocol DNA/RNA extraction of unfed nymphal ticks using QuickExtract (For adult ticks use twice the volume):
1. Mix 50µl water with 50µl QuickExtract.
2. Add to tick homogenate and mix well.
3. Incubate at 65°C for 6 minutes
4. Incubate 95°C for 2 minutes.
5. Cool to room temperature.
6. Store extracts in freezer until use.
CAUTION --Contamination has been detected in samples that were extracted using a DNA extraction robot that was also being used for extracting mouse samples. Bead-beaters may have similar issues. Extracting sham samples and testing for possible contamination sources would be prudent before one processes their irreplaceable field-collected samples.
III. PCR Set-up
SsoFast Probes Supermix (Biorad #172-5230)
Primers (see Table 1 for sequences), suggested supplier is Integrated DNA Technologies
PrimeTime qPCR double-quenched probes from Integrated DNA Technologies (see Table 1 for sequences). Multiplex reactions are run with the fluorophores: FAM, HEX and CY5.
Molecular biology grade water (UltraPure DNase/RNase-free distilled water Invitrogen #10977015
White 96-well reaction plate (Greiner Bio-One #669285). Each PCR machine uses a different type, please check with the manufacturer of your machine, if you are not using a LightCycler 480.
Sealing foil (Roche Diagnostics #04729757001
Snap-cap microcentrifuge tubes (Eppendorf #022364111)
Aerosol barrier pipette tips
Roche LightCycler 480
Container with ice
Pipette basins (Fisherbrand #12-681-507)
Eppendorf PCR-cooler (Daigger Scientific #EF2927C)
Contamination prevention procedures for PCR set-up: PCRs are set up in a room physically separated from the real time PCR machines inside a dedicated PCR dead air hood equipped with UV light decontamination. Quality-controlled molecular biology grade water is used. This water is aliquoted into microfuge tubes that are left open under UV illumination overnight to ensure that no amplifiable DNA is present. Pipettors are dedicated for PCR set-ups only. Pipettors used to assemble the Master Mix should never be used for DNA, instead have a dedicated pipettor used to add the DNA template. Aerosol barrier tips are used for all pipetting. All racks are dedicated for PCR set-ups only and are left under the UV lamps for approximately 6 hours in-between runs. Ice packs are also UV irradiated in the PCR hood before being returned to the freezer. (Do not leave pipettors under the UV lights, the radiation will degrade the plastic. These can be decontaminated with DNA-Away (Fisher #21-236-28)). Wear a clean lab coat designated for PCR set-ups (not the same lab coats used for DNA extractions). Finally, consider what else you have done that day that might potentially cause contamination issues. Have you been working with animal tissues? Have you been in your animal room? Have you passed your mouse cells? Have you spent time in the room with your PCR machines? If the answer to any of these questions is yes, perhaps you should wait and set-up this PCR tomorrow. I set-up PCRs first thing in the morning and do all other lab work afterwards.
-Note- This assay was developed on a Roche LightCycler 480; the settings described here, will pertain to that instrument. As with regular real time PCRs, this assay should work on any real time machine with only minor adjustments. Keep in mind that each machine has a unique set of filters for fluorophore detection. If using a different real time machine be sure to check your manufacturer’s instructions to confirm that the fluorophores used here will work with your machine.
PCR reactions are carried out in white 96-well plates. Set-up is done on ice.
DNA templates are always run in duplicate
Multiplex reactions can be run as designated in Table 1.
1. Don clean gloves and a clean lab coat before handling any PCR reagents.
2. Thaw all reagents for Master Mix at room temperature in PCR hood with overhead light off to avoid unnecessary photobleaching of probes.
3. While reagents are thawing, pull DNA templates out of freezer.
4. Thaw DNA; mix thoroughly and then spin down. Keep these away from the PCR hood while you set-up the plate with Master Mix. DNA templates are always run in duplicate. Be sure to include negative controls.
5. After handling DNA templates, change to clean gloves before assembling Master Mix.
6. Mix together reagents for Master Mix as detailed below. Make enough Master Mix for the number of samples being tested (X2 because every sample is done in duplicate) plus a couple extra for pipetting errors. (For example: 47 samples in duplicate= 94. Add 2 negative controls for a full 96-well plate. Add a few extra for pipetting errors= 102. Therefore, make enough Master Mix for 102 reactions for a full 96-well plate.)
PCR Master Mix for one 15µl reaction:
7.5µl SsoFast Probes Supermix
0.12µl of 25µM working stock for each primer (up to 6 primers)
0.2µl of 25µM working stock for each probe (up to 3 probes)
Xµl water to make 13µl total
7. Vortex well and distribute 13µl into each well of 96-well plate
8. Sit plate on ice.
9. Put all Master Mix reagents back in freezer.
10. Bring the racks with the DNA templates into the hood.
11. Add 2µl of template to each well. Pipette up and down to mix.
12. Seal plate and place in real time PCR machine and run using the following cycling conditions:
98°C 2 minutes
Then 50 cycles of
98°C 8 seconds
58°C 30 seconds with a plate read at each cycle.