Feb 05, 2025
Detection of knockdown resistance mutations in Musca domestica by rhPCR
- 1USDA ARS CMAVE
- Estep Laboratory
Protocol Citation: Alden Estep, Neil Sanscrainte 2025. Detection of knockdown resistance mutations in Musca domestica by rhPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj9dqlx9/v1
Manuscript citation:
Estep, A., Sanscrainte, N., Pagac, A., Geden, C. and Burgess IV, E., 2024. Rapid and cost-effective screening of genetic markers associated with pyrethroid resistance in Musca domestica using RNAse H2 PCR (rhPCR). https://doi.org/10.21203/rs.3.rs-5239153/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: October 25, 2023
Last Modified: February 06, 2025
Protocol Integer ID: 89895
Keywords: Musca domestica, knockdown resistance, RNAse H2 PCR, voltage gated sodium channel, insecticide resistance
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Abstract
This protocol details the step-by-step procedure for assessing Musca domestica knockdown resistance (kdr) mutations using RNAse H2 PCR (rhPCR). This procedure utilizes the specificity of rhPCR, the high throughput of 96-well plate-based sample handling, and the rapidity of SYBR based PCR assays to genotype for critical kdr mutations (D600N, M918T, T929I, and L1014H/F). Compared to existing assays for kdr assessment in M. domestica, these assays reduce expense per sample by ~75% and time by ~80%.
Guidelines
None
Materials
Consumables needed for assays:
| A | B | C | |
| Item | Product Number | Vendor | |
| 96 well cap | 276002 | Thermo | |
| 2ml 96 well homogenization plate | 278743 | Thermo | |
| 2.3-mm-diameter zirconia silica beads | 11079124ZX | BioSpec | |
| tips | |||
| dH2O | |||
| Microamp 384-well plate | 4309849 | AB | |
| Sybr Select Master Mix | 4472919 | Invitrogen | |
| rhPCR F primer* | Integrated DNA technologies | ||
| rhPCR R primer* | Integrated DNA technologies | ||
| RNAse H2 | 11-03-02-02 | Integrated DNA technologies | |
| NFW | AM9937 | AB | |
| tips |
Safety warnings
Always use proper PPE.
Follow all laboratory safety procedures.
Follow all institutional and manufacturer guidelines for the safe and proper use of laboratory equipment and reagents.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
None
Sample preparation
Sample preparation
3m
3m
Room temperature Add cubic zirconium beads to Omni Products 96-deep well plate using Biospec bead loader
Room temperature Add 400 µL of deionized water using Eppendorf Repeatter E3
Clean forceps with 70% ethanol solution
On ice
Note
Save column 1 for addition of controls.
Maintain samples and plates on ice while preparing samples.
Remove 2-3 legs from a single organism and place into well A2. Place carcass into well A2 of a 96-well storage plate.
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | ||||||
| B | ||||||
| C | ||||||
| D | ||||||
| E | ||||||
| F | ||||||
| G | ||||||
| H |
| 7 | 8 | 9 | 10 | 11 | 12 | |
| A | ||||||
| B | ||||||
| C | ||||||
| D | ||||||
| E | ||||||
| F | ||||||
| G | ||||||
| H |
go to step #3 On ice Repeat process of filling plate for columns 2 through 12. Load samples by working down columns rather than across rows. This results in reduced reagent consumption for automated reaction assembly of partial plates.
On ice Using same procedure as in steps 4-6, add controls to Wells A1-H1. Alternatively, purified synthetic DNA can be used as controls.
A1: 1014L allele/918M allele/929T allele/600D allele (no kdr CAR21 strain- Carolina Biological Supply)
B1: 1014H allele/918M allele/929T allele/600D allele (NCHis strain- Jeff Scott Cornell University)
C1: 1014F allele/918M allele/929T allele/600D allele (ALkdr strain- Jeff Scott Cornell University)
D1: 918T allele/929T allele/600D (JPSkdr strain- Jeff Scott Cornell University)
E1: 918M allele/929I allele/600D allele (kdr1b strain- Jeff Scott Cornell University)
F1: 918T allele/929T allele/600N (TypeN strain- Jeff Scott Cornell University)
G1: no sample blank
H1: no sample blank
On ice Seal leg plate with Omni Products silicon sealing mat. Seal carcass plate with foil seal. Freeze carcass plate at -80C to maintain RNA.
Homogenize sealed leg plate for 1 min at 30 hertz on Omni plate homogenizer.
00:01:00
Equipment
BEAD RUPTOR 96
NAME
homogenizer
TYPE
Omni International
BRAND
27-0001
SKU
1m
2000 rpm, Room temperature, 00:02:00
Equipment
Centrifuge
NAME
Eppendorf
BRAND
5804
SKU
Centrifuge leg sample plate.
2m
On ice Return plate to ice.
Reagent Preparation
Reagent Preparation
3m
3m
Label 9 - 1.7ml microcentrifuge tubes (as 1-9) to prepare allele specific rhPCR reactions.
On ice Prepare 300 ul of 20 mU/ul H2 enzyme by diluting 3ul of 2U/ul H2 enzyme in 297ul of H2 buffer (IDTDNA).
Prepare 9 allele-specific H2 PCR reactions following the table below:
Note
Primers and H2 enzyme can be ordered from IDT DNA. SYBRGreen is from ThermoFisher.
All values are in microliters.
| Reaction tube #: | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
| Allele: | 1014L | 1014H | 1014F | 918M | 918T | 929T | 929I | 600D | 600N | |
| SYBR | 560.00 | 560.00 | 560.00 | 560.00 | 560.00 | 560.00 | 560.00 | 560.00 | 560.00 | |
| NFW | 283.36 | 312.48 | 283.36 | 320.32 | 320.32 | 283.36 | 283.36 | 319.76 | 319.76 | |
| 20mU H2 | 43.68 | 14.56 | 43.68 | 6.72 | 6.72 | 43.68 | 43.68 | 7.28 | 7.28 | |
| rhPCR primer: 1014L | 4.48 | |||||||||
| rhPCR primer: 1014H | 4.48 | |||||||||
| rhPCR primer: 1014F | 4.48 | |||||||||
| rhPCR primer: 1014r | 4.48 | 4.48 | 4.48 | |||||||
| rhPCR primer: 918M | 4.48 | |||||||||
| rhPCR primer: 918T | 4.48 | |||||||||
| rhPCR primer: 918r | 4.48 | 4.48 | ||||||||
| rhPCR primer: 929T | 4.48 | |||||||||
| rhPCR primer: 929I | 4.48 | |||||||||
| rhPCR primer: 929r | 4.48 | 4.48 | ||||||||
| rhPCR primer: 600D | 4.48 | |||||||||
| rhPCR primer: 600N | 4.48 | |||||||||
| rhPCR primer: 600r | 4.48 | 4.48 |
Seal tubes, vortex briefly to mix and then spin.
Place V-bottom PCR plate into Eppendorf plate rack. Aliquot 108ul of reaction 1 to each well of column 1.
go to step #15 Repeat step above for reactions 2-9.
Reaction Assembly on Eppendorf 5750
Reaction Assembly on Eppendorf 5750
3m
3m
Start Eppendorf 5750 system.
Load (or write) program USDA/Musca domestica/20210925.
Note
This program aliquots each of the 9 allele-specific mastermixes into 384-well plates and then adds homogenate from the homogenized sample plate.
Program is as follows:
Setup platform of Ep5750 workstation as shown below.
Place tips in positions A2, A3 and B2.
Place labelled 384-well plates in positions C2, C3 and C4.
Place aliquoted reagent plate in B3.
Place homogenized sample plate in B4.
Close safety cover of Ep5750 workstation.
Start program and allow to run until reaction setup is complete.
Exit Ep5750 program and open cover.
Seal 384-well plates with Eppendorf Optical covers. Seal tightly around edges with sealing tool.
Spin plates for 30 seconds in plate centrifuge and place covered in refrigerator until amplification.
Clean Ep5750 worksurface and shutdown workstation.
Remove empty tip racks.
Recover partial tip rack.
Remove sample homogenate plate from Ep5750 platform and reseal with silicone sealing mat. Freeze immediately at -80C to preserve RNA and reduce degradation.
Empty platform waste container.
Wipe platform with a water moistened paper towel.
Shutdown workstation computer and switch off Ep5750.
Thermocycling procedure
Thermocycling procedure
3m
3m
Start Applied Biosystems QuantStudio6 Flex and open QuantStudio software
Select Open/Template/Mdom15State on Desktop
Open Mdom_Plate_1.edt, Mdom_Plate_2.edt and Mdom_Plate_3.edt
Add sample information and date to comments box for plates 1,2 and 3
Save each as a .eds file starting with date of test as:
YYYYMMDD_Mdom_Plate_1
Extend plate carrier by selecting front panel virtual door button.
Load Plate_1 ensuring position A1 is at upper left as marked on plate holder.
Close door by pressing virtual door button on front panel.
01:04:00 Press start and allow approximately 64 minutes to complete run
1h 4m
go to step #28.3 Repeat substeps for Plate_2 and Plate_3
Data analysis
Data analysis
Note
Allele presence or absence for each sample can be done by visual inspection of the amplification curves directly in the QuantStudio6 software or by exporting an excel file and calling alleles based on cycling threshold values and melting temperature. This procedure details the visual inspection method for the presence or absence of the 1014L allele. Calls for the other 8 reactions are done the same way.
Assess overall assay validity by examining Wells M1 and O1. They should not amplify as they contain no template. Amplification indicates contamination and any assay with amplification in blank wells should be redone.
Select controls with known 1014L alleles (CAR21 in position A1) and control without 1014L alleles (C1, E1, G1, I1, K1). The 1014L positive samples will amplify within a small range of cycles and the 1014L negative samples will appear much later (note that they will usually appear with enough cycling time).
Examine each test samples to determine whether it is positive or negative for an allele based on the controls.
Note
Some results may be indeterminate (amplify between clear positive and negative groups) and will need to be rerun or verified with another method.
go to step #29.1 Repeat step 29 for each of the remaining 8 alleles.
Protocol references
Freeman, J. C., Ross, D. H. & Scott, J. G. Insecticide resistance monitoring of house fly populations from the United States. Pestic Biochem Physiol 158, 61-68 (2019). https://doi.org/10.1016/j.pestbp.2019.04.006
Mertz, R. W. et al. Frequencies and distribution of kdr and Ace alleles that cause insecticide resistance in house flies in the United States. Pesticide Biochemistry and Physiology 194, 105497 (2023).
Sun, H., Tong, K., Kasai, S. & Scott, J. Overcoming super‐knock down resistance (super‐kdr) mediated resistance: multi‐halogenated benzyl pyrethroids are more toxic to super‐kdr than kdr house flies. Insect molecular biology 25, 126-137 (2016).
Dobosy, J. R. et al. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide
polymorphism detection using blocked cleavable primers. BMC Biotechnol 11, 80 (2011). https://doi.org/10.1186/1472-6750-11-80
Nakauchi, M. et al. Rapid detection of an I38T amino acid substitution in influenza polymerase acidic subunit associated with reduced susceptibility to baloxavir marboxil. Influenza Other Respir Viruses 14, 436-443 (2020). https://doi.org/10.1111/irv.12728
Barefield, D. Y., Lynch, T. L. t., Jagadeesan, A., Sanagala, T. & Sadayappan, S. High-Throughput Diagnostic Assay for a Highly Prevalent Cardiomyopathy-Associated MYBPC3 Variant. J Mol Biomark Diagn 7 (2016). https://doi.org/10.4172/2155-9929.1000303
Labbe, G. et al. Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR. J Microbiol Methods 157, 81-87 (2019). https://doi.org/10.1016/j.mimet.2018.12.021