TITLE: YDV Multiplex PCR
AUTHORS: Stephen Byrne, Virgile Ballandras, Louise McNamara

[DESCRIPTION]
This protocol describes the process of carrying out a multiplex PCR assay followed by capillary electrophoresis to detect C/BYDV and determine species (BYDV-MAV, BYDV-PAS, BYDV-PAV, CYDV (RPS or RPV)). The starting point for this is cDNA that has been synthesized from nucleic acid extracted from single aphids or leaf material - an internal control targeting GAPDH is  included and primers can be selected depending on the sample matrix (aphid or barley). The BYDV primers were taken from:
 
Sõmera, M., Massart, S., Tamisier, L., Sooväli, P., Sathees, K. and Kvarnheden, A., 2021. A survey using high-throughput sequencing suggests that the diversity of cereal and barley yellow dwarf viruses is underestimated. Frontiers in Microbiology, 12, p.673218.
 
In-house sequence data was used to modify primers where necessary, add a primer for CYDV and an internal control for both aphid and barley samples. The CYDV primers captures both RPS and RPV (high resolution melt analysis may be required to distinguish these if required). 

[BEFORE_START]
This protocol assumes that you have already isolated Total RNA from plant or insect material, and converted to cDNA using random hexamers.  The protocol uses capillary electrophoresis for separation of PCR products; however it is also possible to separate and visualise using a high quality agarose gel electrophoresis method.  

[STEPS]
SECTION: PCR protocol
1. Identify samples for analysis, including positive (virus infected aphid/barley) and negative controls (virus free aphid/barley), a positive control consisting of a mix of gBlocks with sequences for all targets in the multiplex PCR, and a no-template control.  Details on the gBlocks can be found here:     

SECTION: PCR protocol
3. Prepare a master-mix for the number of samples to be run so that each well will contain:

 12.5 µL  of    
10.5 µL of molecular grade nuclease-free water
1 µL of working solution of appropriate primer mix prepared in step 2 above

 


SECTION: PCR protocol
2. Prepare the primer mix for the multiplex PCR (YDVmixHV when sample matrix is barley, or YDVmixSA when sample matrix is an aphid) using primers according to tables below. The primer mixes are prepared with each primer in the working solution at  10 micromolar (µM) .

YDVmixHV:

 
 YDVmixSA:

 
 
 
SECTION: PCR protocol
4. Pipette 1 µL  of cDNA (or control) into each well of a PCR plate and keep on ice.

SECTION: PCR protocol
5. Pipette 24 µL of master-mix from step 3 into each well, seal the plate carefully with foil lid, and centrifuge the PCR plate briefly to ensure no droplets adhere to sides of sample wells. 

SECTION: PCR protocol
6. Place the PCR plate in a 
 
and run the following PCR cycle:

95 °C 5 min 

followed by 40 cycles of

 95 °C 30 s 
 60 °C 90 s 
 72 °C 30 s 

and a final elongation step of

 68 °C 10 min 

SECTION: Capillary Electrophoresis
7. Bring all reagents from dsDNA reagent kit (1-500 bp; DNF-905) to room temperature by removing from the fridge and freezer at least 30 min prior to setting up a run.

SECTION: Capillary Electrophoresis
8. Set up a run on the 5200 Fragment Analyser by adding 1 mL of 1X Inlet Buffer into row A of the 1.2 ml deep 96-well plate (to make 12 mL fresh add 2.4 mL of 5X Inlet buffer to 9.6 mL of molecular grade water. Inlet buffer should be changed daily. Into the same plate place 1 mL  of Capillary Storage solution into row H (note: the storage buffer only needs to be changed on a weekly basis). This plate needs to go into drawer "B" of the instrument. 

SECTION: Capillary Electrophoresis
9. Add 30 µL of ready to use marker (1 and 500 bp) to row A of a compatible 96-well PCR plate and add a drop of miner oil (provided in kit) to each well and place plate into drawer "M" of the instrument. The marker is good for 30+ injections or 1 month.

SECTION: Capillary Electrophoresis
10. Prepare sufficient 1X conditioning solution for the number of samples you are going to run (e.g. 45 mL is sufficient for a single plate) and connect to correct fluid line.

SECTION: Capillary Electrophoresis
11. Prepare sufficient gel for the number of samples you are going to run (e.g. 4.5 µL added to 45 mL  of separation gel is sufficient for a single plate) and connect to correct fluid line.

SECTION: Capillary Electrophoresis
12. The samples are prepared by adding 2 µL of PCR product to 22 µL of 1X TE (provided) in the well of a compatible PCR plate. Vortex the plate and centrifuge briefly to collect contents at bottom of the wells.

SECTION: Capillary Electrophoresis
13. Update the solution levels on instrument and commence a run using method appropriate for the DNF-905-dsDNA (1 to 500 bp) kit. 
SECTION: Capillary Electrophoresis
14.