Dec 13, 2024
Multiplex PCR for Tetracycline Resistance Genes SOP
- Justine C. Condon1,
- Lisa M. Durso1
- 1USDA-ARS
Protocol Citation: Justine C. Condon, Lisa M. Durso 2024. Multiplex PCR for Tetracycline Resistance Genes SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzn61lzp/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: September 25, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 108737
Keywords: PCR Tracker, Gel electrophoresis , gBlock, Antibiotic Resistance, Tetracycline Resistance, Polymerase Chain Reaction, PCR, Environmental, Soil, Water, Agroecosystem, Agriculture
Funders Acknowledgements:
USDA-ARS
Grant ID: NP 212 Soil and Air
Disclaimer
The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.
Abstract
The tetracyclines are the most widely used drugs in food animal production both globally, and in the United States (U.S.). They account for 36% of all drugs sold in the U.S. in 2018 for veterinary purposes, with 44% of those attributed to use in cattle and 48% in swine. Although these drugs are classified as “Highly Important” (the second category) by the World Health Organization (WHO), they are not listed on the Center for Disease Control's (CDC) 2019 Antibiotic Resistance Threats Report. However, they are commonly used to assess antibiotic resistance in soil and water. This protocol describes the correct procedures on how to set-up and run the Tetracycline Gene Multiplex PCR assay.
Image Attribution
Stock Photo
Guidelines
Scope:
Tetracycline is an antibiotic used to treat a broad spectrum of infections including types of pneumonia, acne, chlamydia, certain types of diarrhea, and some infections carried by ticks. While commonly used in humans, tetracyclines are sometimes used to treat pets, and widely used for food animals. Antibiotic drugs are used to treat infectious diseases, however over time the bacteria that cause the diseases have grown resistant to the drugs. When this happens people and their pets are sick longer or may even die where once they would have survived. Just as there are many different types of antibiotic drugs, there are also many types of antibiotic resistance. This protocol describes how to tell if a bacteria has the potential to be resistant to tetracycline drugs, and describes an assay that measures multiple tetracycline resistance genes at the same time.
Responsibility:
This SOP applies to all staff members and students. These individuals must be knowledgeable about the requirements set forth within this document. The lab manager or designee shall ensure that all staff and students know the proper techniques.
Disclaimer:
The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.
Materials
Reagents and Materials:
- JumpStart™ REDTaq® ReadyMix™ Reaction MixMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0982
- Forward and Reverse Primers – See Table 2 (Step 4)
- PCR Water (HyClone SH30538.01 Sigma, St. Louis, MO)
- Positive Controls - gBlocks 9-15 - See Table 3 (Step 10) and Appendix (Steps 31-37)
- AgaroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9539
- SYBR SAFE DNA stainInvitrogen - Thermo FisherCatalog #S33102
- DirectLoad™ PCR 100 bp Low LadderMerck MilliporeSigma (Sigma-Aldrich)Catalog #D3687
- 0.2 mL x 8 PCR reaction strips (T320-2N, Simport, Quebec, Canada)
- 2 mL sterile centrifuge tube (02.681.299 Fisher Scientific, Pittsburg, PA)
- Aerosol barrier no retention pipette tips (Various)
- 1X SB buffer (20X Sodium Borate (SB) Buffer - SB20-4, FasterBetter Media, Hunt Valley, MD)
Equipment:
- PCR Prep Cabinet (3620804 Labconco, Kansas City, MO)
- Pipettors for various volumes from 1-1000µl (Gilson, Middleton, WI)
- Thermal cycler (T100, BioRad, Hercules, CA)
- Mini Centrifuge (C1301P, Labnet International, Big Flats, NY)
5. 500ml or 1-liter sterile Pyrex glass bottle
6. Gel casting tray, gel combs, and gel boat
7. Electrophoresis machine (PowerEase 300W, Life Technologies, Carlsbad, CA)
8. Gel imaging apparatus (Gel Doc XR+, BioRad, Hercules, CA)
9. Lighter
Safety warnings
Wear proper personal protective equipment (PPE) when working through PCR: nitrile gloves and lab coat.
When working with the hot agarose, wear thermal hot-pad gloves.
Beware, overmixing can degrade DNA.
Procedure – PCR
Procedure – PCR
15m 20s
15m 20s
If frozen, thaw all reagents and samples and keep On ice or ice block for stability. If kept in the refrigerator, keep there until ready for use, and keep On ice or ice block for stability when in use.
Label tubes for the samples and controls.
Prepare the PCR prep area for setting up a PCR reaction to eliminate contamination – UV 0.2mL 8-strip PCR tubes, PCR water, and microcentrifuge tube for 00:15:00 .
15m
Combine the Master Mix components for the number of reactions (allow for 10% extra Master Mix) using the recipe from Table 1 with the correct primer sets (Table 2) for 25 µL reactions. Before adding to the Master Mix - mix the primers by aspirating and expelling with the pipette tip.
| A | B | C | D | |
| Ingredient | Source | Volume used (µL) | Final Concentration | |
| MasterMix | JumpStart Red Taq Ready Mix (P0982-800rxn, Sigma) | 12.5 | 1X | |
| PCR Water | HyClone (SH30538.01, Sigma) | 5.5 (or 4.7 for PQX) | N/A | |
| Forward Primer 1 | 2-3 primers (100µM) | 0.5 | 2.0µM | |
| Reverse Primer 1 | 2-3 primers (100µM) | 0.5 | 2.0µM | |
| Forward Primer 2 | 2-3 primers (100µM) | 0.5 (0.4 for Tet X) | 2.0µM | |
| Reverse Primer 2 | 2-3 primers (100µM) | 0.5 (0.4 for Tet X) | 2.0µM | |
| Sample (template) | 1µL/rxn | 5 | Varies |
Table 1. PCR recipe for a 25µL reaction using 5µL of sample.
Table 2. Tetracycline multi-plex groupings including its primer sequences, thermal cycler conditions, and amplicon size.
Vortex the completed Master Mix for at least 00:00:20 .
20s
*Check the pipette’s volume as you go along, making sure it is exactly at the correct volume*
Carefully pipette 5 µL of PCR Water to the non-template control (NTC) tube.
Add 20 µL of Master Mix to each labeled 0.2mL PCR strip tube.
Next, gently vortex to mix, then pipette 5 µL of sample (5µl of working stock; or 1µl straight DNA + 4µl of PCR water) into its same labeled tube.
Note
Use a new pipette tip for each sample.
To make a working stock dilution see SOP: IDT Primer and gBlock Hydration and Aliquots.
Add 1 µL of positive control (Table 3) (mix first by aspirating and expelling with the pipette tip) to its same labeled tube.
Note
The correct concentration of gBlock (106) should already be pre-made and stored at -20oC.
| A | B | C | |
| Lab ID | Primer 1 | Primer 2 | |
| gB9-TetW | P37 -Tet W | N/A | |
| gB10-Tet B-X | P15-Tet B | P27-Tet X | |
| gB11-Tet C-P | P16 - Tet C | P24 - TetP | |
| gB12-Tet A-Q | P14 - Tet A | P25 - Tet Q | |
| gB13-Tet G-O | P19- Tet G | P23 - Tet O | |
| gB14-Tet E-M | P18 - Tet E | P22 - Tet M | |
| gB15-Tet D-L | P17 - Tet D | P21 - Tet L |
Table 3. Tetracycline positive control gBlock ID numbers and associated primers.
Centrifuge the 0.2mL PCR strip tubes so that the entire volume is at the bottom.
Note
Make sure the centrifuge is balanced with both sides containing tubes.
Load the tubes into the Thermocycler, keeping them in order.
Find the programmed gene conditions listed under ‘Tet_ --’ and double check that they match the conditions listed in Table 2.
Table 2. Tetracycline multi-plex groupings including its primer sequences, thermal cycler conditions, and amplicon size.
Run PCR
When cycle is complete:
- Gel electrophoresis (Section Procedure – Gel Electrophoresis) OR
- Place the tubes in a labeled rack at -20oC until ready to run a gel
- Turn off the Thermocycler.
Procedure – Gel Electrophoresis
Procedure – Gel Electrophoresis
3h 11m 30s
3h 11m 30s
Using a spoon, measure out 2.6 g of powdered agarose into a weigh boat.
Measure 260 mL of 1X SB buffer into a graduated cylinder.
Add the powdered agarose and 1X SB into 500mL bottle and swirl to mix.
Microwave the SB and agarose mixture for 00:01:00 with bottle lid LOOSE.
1m
Remove bottle from microwave (using hot pad gloves), tighten the lid and gently swirl.
Safety information
For Steps 19 - 27 - Use thermal hot pad gloves to protect from burns.
LOOSEN the lid and put in microwave for another minute.
Check mixture visually, if flecks are present repeat go to step #19 , loosen lid and microwave again for 00:00:30 .
30s
Add 26 µL of SYBR safe to the molten gel.
Swirl gently with a loose lid to distribute the SYBR.
Place bottle in a 55 °C water bath for 00:10:00 to temper.
10m
While the mixture is tempering, set up gel cast by putting two plastic stoppers into the ends of the cast, with the orange rubber side of the stopper facing out.
Put two (or three) 36-well combs into the first and fourth (or first, third, fifth) notches of the gel cast (gel cast is right side up if first notch is close to the top of the cast).
After the gel has tempered, slowly pour it (using hot pad gloves) into the gel cast.
If there are any noticeable bubbles, take a lighter and hold it over the bubble, which should make it pop.
Cover gel loosely with foil and allow it to set before use (up to 01:00:00 ).
Note
**SB gels will be flimsier, so be careful when removing the combs or transporting the gel**
1h
Electrophoresis Conditions
10 µL of PCR amplicon will go into a well or 5 µL of ladder.
Run at 120 V for 02:00:00 in 1x SB buffer.
Note
Make sure SB gel goes into a gel boat with SB buffer.
2h
Remove the processed gel and analyze using a gel imaging system.
Appendix – gBlock Sequences
Appendix – gBlock Sequences
*Note: 'Reverse' denotes the 'Reverse Complement'
gBlock 9 – Tet W (228 bp) - for Tet assay and primer set P37 – Tet W
gBlock 10 – Tet B-X (1150 bp) - for Tet assay and primer set P15 – Tet B, and P27 – Tet X
gBlock 11 – Tet C-P (1109 bp) - for Tet assay and primer set P16 – Tet C, and P24 – Tet P
gBlock 12 – Tet A-Q (1239 bp) - for Tet assay and primer set P14 – Tet A, and P25 – Tet Q
gBlock 13 – Tet G-O (1423 bp) - for Tet assay and primer set P19 – Tet G (Retired Primer Set) and P23 – Tet O
gBlock 14 – Tet E-M (699 bp) - for Tet assay and primer set P18 – Tet E, and P22 – Tet M
gBlock 15 – Tet D-L (1069 bp) - for Tet assay and primer set P17 – Tet D (Retired Primer Set) and P21 – Tet L
Protocol references
Aminov et al., 2001 Aminov, R.I., Garrigues-Jeanjean, N., Mackie, R.I. Molecular Ecology of Tetracycline Resistance: Development and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins. AEM (2001) 67:1 22-32.
CDC. Antibiotic Resistance Threats in the United States, 2019. Atlanta, GA: U.S. Department of Health and Human Services, CDC; 2019. 2019 Antibiotic Resistance Threats Report | Antimicrobial Resistance | CDC
Ng, L.-K., et al. “Multiplex PCR for the Detection of Tetracycline Resistant Genes.” Molecular and Cellular Probes, vol. 15, no. 4, 15 Jan. 2001, pp. 209–215, https://doi.org/10.1006/mcpr.2001.0363.
Acknowledgements
A thank you to Ryan McGhee (USDA-ARS) and Dr. Lana Castleberry (USDA-ARS) for their assistance in getting this assay up and running.