TITLE: Molecular testing of carrion flies for rabbit calicivirus detection
AUTHORS: Robyn Hall

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Carrion fies are relatively easy and inexpensive to collect, systematic sampling networks can be established, and rabbit calicivirus can be detected in flies using high-sensitivity molecular methods. However, previous studies were  conducted when there was only a single pathogenic rabbit calicivirus circulating in Australia. The aim of this study was to optimise molecular testing protocols of carrion flies for rabbit calicivirus detection at the virus variant level. </div></div>

[STEPS]
?. [Trapping of flies]
Ensure trap has been thoroughly decontaminated by soaking in 10% household bleach for at least 30 minutes, followed by rinsing with water. Place attractant in a specimen jar covered with a gauze swab to prevent flies coming into direct contact with the bait.Place trap in collection location for one to seven days. Freeze entire trap at  to immobilise flies. Transfer >48 mg of fly tissue (approximately five large calliphorid flies (e.g. C. augur, C. stygia) or seven small flies (e.g. Chrysomya varipes) to a pre-weighed 1.8 ml tube containing 1mm glass beads. Negative control flies should be processed in parallel for each batch  of testing.Transfer remaining flies to storage containers for long-term storage at  .Decontaminate trap before reuse.Freeze entire trap at to immobilise flies. Transfer >48 mg of fly tissue (approximately five large calliphorid flies (e.g. C. augur, C. stygia) or seven small flies (e.g. Chrysomya varipes) to a pre-weighed 1.8 ml tube containing 1mm glass beads. Negative control flies should be processed in parallel for each batch of testing.Transfer remaining flies to storage containers for long-term storage at .
?. [Preparation of cartridge for extraction]
Snap cartridges into position on deck tray of the Maxwell RSC instrument. Centre the cartridges in the tray.Remove foil from cartidges. Place an LEV Plunger in well #8 of each cartridge. Add  of DNase I to well #4 of each cartridge. Place elution tube into position on the deck tray.Add  of homogenate/lysis buffer mix to well #1. Place deck tray into Maxwell RSC instrument and open lids of elution tubes. Run Maxwell RSC instrument.Add of DNase I to well #4 of each cartridge. Place elution tube into position on the deck tray.Add  of homogenate/lysis buffer mix to well #1. Place deck tray into Maxwell RSC instrument and open lids of elution tubes. Run Maxwell RSC instrument.Add of homogenate/lysis buffer mix to well #1. Place deck tray into Maxwell RSC instrument and open lids of elution tubes. Run Maxwell RSC instrument.Immediately after extraction, store RNAs at . Dispose of chemical waste in accordance with local guidelines.
10 µl
400 µl
?. [RT-qPCR]
As described in https://www.ncbi.nlm.nih.gov/pubmed/29226567Kit: SensiFAST™ SYBR® No-ROX Kit (Bioline)Primers: GI_qRTPCR_Fw (5’- TTGACRTACGCCCTGTGGGACC-3’)GI_qRTPCR_Rv (5’- TCAGACATAAGARAAGCCATTRGYTG-3’)Templates: NTC (nfH2O) in duplicate, RHDV standards in duplicate (101 to 107), fly RNAs in duplicate, positive control in duplicate  AB1ReagentAmount (ul)2nfH2O2.732x OneStep mix5410 uM GI_qRTPCR_Fw0.5510 uM GI_qRTPCR_Rv0.56RNase inhibitor0.27Reverse transcriptase0.1Add of mastermix to respective wells of a 96 well PCR plate.Add of RNA template to respective wells. Include RHDV standards for quantification (see publication for details), a no template control, +/- a positive control to monitor interrun variation.Cycling: for for 40 cycles of:for for for with acquisitionMelt curve to with an increment of and acquisition
AB1ReagentAmount (ul)2nfH2O2.732x OneStep mix5410 uM GI_qRTPCR_Fw0.5510 uM GI_qRTPCR_Rv0.56RNase inhibitor0.27Reverse transcriptase0.1
9 µl
1 µl
?. [Preparation for RNA extraction]
Decontaminate work area prior to commencing RNA extraction. Ensure DNase I has been prepared by adding  of nuclease-free water and  of Blue Dye to one vial of lyophilised DNase I. Store at  .Set heat-block to  .To avoid contaminating stock bottles, prepare aliquots of a) homogenisation buffer containing 20 µl.ml-1 of 1-Thioglycerol (chill before use), b) lysis buffer, and c) DNase I.Ensure DNase I has been prepared by adding of nuclease-free water and of Blue Dye to one vial of lyophilised DNase I. Store at .Set heat-block to .Label one 0.5 ml elution tube per sample and add of nuclease-free water to the bottom of each tube.Add  of homogenisation buffer containing 1-Thioglycerol per mg of dry fly tissue volumes (minimum 200 μl).Heat samples at for  and allow to cool for .Mix homogenate (minimum 200 μl) with an equal volume of lysis buffer by vortexing for .
275 µl
5 µl
50 µl
10 µl
?. [Freeze-drying]
Loosen lids on tubes containing flies and place in freeze-drying chamber.Freeze-dry overnight and weigh tube after freeze-drying to calculate dry fly tissue weight.Homogenise samples using a Precellys 24-dual tissue homogeniser (Bertin technologies). Spin homogenised samples briefly to collect material at the bottom of the tube.
?. [Strain-specific RT-PCR]
As described in https://www.ncbi.nlm.nih.gov/pubmed/29226567. Kit: OneStep Ahead RT-PCR Kit (Qiagen)Primers:  ABCD1Primer nameSequence (5′→3′)Amplicon size (bp)Binding region2GI.1a‐Aus_fwdGCGTGGCATTGTGCGCAGCATC562Non-structural3GI.1a‐Aus_revTGTTGGTGATAAGCCATAATCGCG4GI.1c_fwdAGCAAGACTGTTGACTCAATTTCG435Capsid5GI.1c_revAGGCCTGCACAGTCGTAACGTT6GI.2_fwdTTTCCCTGGAAGCAGTTCGTCA336Capsid7GI.2_revTGTTGTCTGGTTTATGCCATTTGC8GI.1a‐K5_fwdTTTATAGATGTATGCCCGCTCAAC263Non-structural9GI.1a‐K5_revCCGTTCGAGTTCCTTGCGGACGTemplates: NTC (nfH2O), fly RNA, strain-specific positive controlDilute RNAs 1/10 in nuclease-free water.Prepare four RT-PCR mastermixes - one per primer pair.  Add  of mastermix to respective PCR tubes.Add  of diluted RNA template to respective tubes.Cycling:  for   for  40 cycles of: for   for   for  Final extension at  for  Hold at   Run PCRs on a 1-1.5% agarose gel at 80-100 V for 30-60 minutes.   AB1ReagentAmount (ul)2nfH2O3.63OneStep Ahead RT-PCR Master Mix4.04OneStep Ahead RT Mix0.4510 uM Fwd primer0.5610 uM Rev primer0.5Add of mastermix to respective PCR tubes.Add of diluted RNA template to respective tubes.Cycling: for for 40 cycles of:for for for Final extension at for Hold at
ABCD1Primer nameSequence (5′→3′)Amplicon size (bp)Binding region2GI.1a‐Aus_fwdGCGTGGCATTGTGCGCAGCATC562Non-structural3GI.1a‐Aus_revTGTTGGTGATAAGCCATAATCGCG4GI.1c_fwdAGCAAGACTGTTGACTCAATTTCG435Capsid5GI.1c_revAGGCCTGCACAGTCGTAACGTT6GI.2_fwdTTTCCCTGGAAGCAGTTCGTCA336Capsid7GI.2_revTGTTGTCTGGTTTATGCCATTTGC8GI.1a‐K5_fwdTTTATAGATGTATGCCCGCTCAAC263Non-structural9GI.1a‐K5_revCCGTTCGAGTTCCTTGCGGACG
AB1ReagentAmount (ul)2nfH2O3.63OneStep Ahead RT-PCR Master Mix4.04OneStep Ahead RT Mix0.4510 uM Fwd primer0.5610 uM Rev primer0.5
9 µl
1 µl