TITLE: Detection of knockdown resistance mutations in Musca domestica by rhPCR
AUTHORS: Alden Estep, Neil Sanscrainte

[DESCRIPTION]
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%.

[BEFORE_START]
None

[GUIDELINES]
None

[STEPS]
SECTION: Reagent Preparation
11. Label 9 - 1.7ml microcentrifuge tubes (as 1-9) to prepare allele specific rhPCR reactions.
SECTION: Sample preparation
1. Room temperature Add cubic zirconium beads to Omni Products 96-deep well plate using Biospec bead loader

SECTION: Sample preparation
2. Room temperature Add 400 µL of deionized water using Eppendorf Repeatter E3

SECTION: Sample preparation
3. Clean forceps with 70% ethanol solution
SECTION: Sample preparation
4. on ice 
 
Remove 2-3 legs from a single organism and place into well A2. Place carcass into well A2 of a 96-well storage plate.
 


SECTION: Sample preparation
5.   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.

SECTION: Sample preparation
7. 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.

SECTION: Sample preparation
6. 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
SECTION: Sample preparation
8. Homogenize sealed leg plate for 1 min at 30 hertz on Omni plate homogenizer.
1 min 
 


SECTION: Sample preparation
9. 2000 rpm, 2 min 
 
Centrifuge leg sample plate.

SECTION: Sample preparation
10. on ice Return plate to ice.

SECTION: Reagent Preparation
12. 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).

SECTION: Reagent Preparation
13. Prepare 9 allele-specific H2 PCR reactions following the table below:

 


 
 
 
SECTION: Reagent Preparation
15. Place V-bottom PCR plate into Eppendorf plate rack. Aliquot 108ul of reaction 1 to each well of column 1.


SECTION: Reagent Preparation
14. Seal tubes, vortex briefly to mix and then spin.
SECTION: Reagent Preparation
16.   Repeat step above for reactions 2-9.

SECTION: Reaction Assembly on Eppendorf 5750
19. 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.
 

SECTION: Reaction Assembly on Eppendorf 5750
17. Start Eppendorf 5750 system. 
SECTION: Reaction Assembly on Eppendorf 5750
18. Load (or write) program USDA/Musca domestica/20210925.

 
Program is as follows:

 

 

SECTION: Reaction Assembly on Eppendorf 5750
20. Close safety cover of Ep5750 workstation.
SECTION: Reaction Assembly on Eppendorf 5750
21. Start program and allow to run until reaction setup is complete.

SECTION: Reaction Assembly on Eppendorf 5750
22. Exit Ep5750 program and open cover.
SECTION: Reaction Assembly on Eppendorf 5750
23. Seal 384-well plates with Eppendorf Optical covers. Seal tightly around edges with sealing tool.
SECTION: Reaction Assembly on Eppendorf 5750
24. Spin plates for 30 seconds in plate centrifuge and place covered in refrigerator until amplification.

SECTION: Reaction Assembly on Eppendorf 5750
25. Clean Ep5750 worksurface and shutdown workstation.

SECTION: Reaction Assembly on Eppendorf 5750
25.1. Remove empty tip racks. 
SECTION: Reaction Assembly on Eppendorf 5750
25.2. Recover partial tip rack.
SECTION: Reaction Assembly on Eppendorf 5750
25.3. Remove sample homogenate plate from Ep5750 platform and reseal with silicone sealing mat. Freeze immediately at -80C to preserve RNA and reduce degradation.
SECTION: Reaction Assembly on Eppendorf 5750
25.4. Empty platform waste container.
SECTION: Reaction Assembly on Eppendorf 5750
25.5. Wipe platform with a water moistened paper towel.
SECTION: Reaction Assembly on Eppendorf 5750
25.6. Shutdown workstation computer and switch off Ep5750.
SECTION: Thermocycling procedure
26. Start Applied Biosystems QuantStudio6 Flex and open QuantStudio software
SECTION: Thermocycling procedure
27. Select Open/Template/Mdom15State on Desktop
SECTION: Thermocycling procedure
28. Open Mdom_Plate_1.edt, Mdom_Plate_2.edt and Mdom_Plate_3.edt
SECTION: Thermocycling procedure
28.1. Add sample information and date to comments box for plates 1,2 and 3

SECTION: Thermocycling procedure
28.2. Save each as a .eds file starting with date of test as:
YYYYMMDD_Mdom_Plate_1
SECTION: Thermocycling procedure
28.3. Extend plate carrier by selecting front panel virtual door button.
SECTION: Thermocycling procedure
28.4. Load Plate_1 ensuring position A1 is at upper left as marked on plate holder.
SECTION: Thermocycling procedure
28.5. Close door by pressing virtual door button on front panel.
SECTION: Thermocycling procedure
28.6. 64 min Press start and allow approximately 64 minutes to complete run

SECTION: Thermocycling procedure
28.7.   Repeat substeps for Plate_2 and Plate_3

SECTION: Data analysis
29. 
 

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.

SECTION: Data analysis
29.1. 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). 
 

SECTION: Data analysis
29.2. Examine each test samples to determine whether it is positive or negative for an allele based on the controls. 
 

SECTION: Data analysis
29.3.   Repeat step 29 for each of the remaining 8 alleles.