TITLE: Rabbit calicivirus capsid Taqman RT-qPCR
AUTHORS: Robyn Hall, Lily Tran, Nina Huang, Ina Smith, Tanja Strive

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To improve the sensitivity and throughput of molecular testing for rabbit caliciviruses we have developed a multiplex TaqMan RT-qPCR assay that detects the three different pathogenic rabbit calicivirus capsid genotypes currently circulating in Australia. Target sequences for all three variants (GI.1c, GI.2, GI.1a) are located within the VP60 capsid sequence. This will also detect subgenomic RNAs, increasing the sensitivity of the method when compared to assays targeting the non-structural gene sequences. Currently this assay is designed to be run as a one-step RT-qPCR assay for high throughput, rapid diagnostic purposes. It has not been designed for quantification of virus loads. For that purpose, the universal lagovirus SYBR-green assay is still recommended (Hall et al, 2018). This assay has not been designed for the detection of recombinant viruses, and will only return the capsid variant/s present in a sample. </div></div>

[STEPS]
?. [Plate set-up]
ABCDEFGHIJKL1GI.1GI.1RNA 6RNA 6RNA 15RNA 15RNA 24RNA 24RNA 33RNA 33RNA 41RNA 412GI.2GI.2RNA 7RNA 7RNA 16RNA 16RNA 25RNA 25RNA 34RNA 34RNA 42RNA 423GI.1aGI.1aRNA 8RNA 8RNA 17RNA 17RNA 26RNA 26RNA 35RNA 35RNA 43RNA 434  NTC    NTC  RNA 9RNA 9RNA 18RNA 18RNA 27RNA 27RNA 36RNA 36RNA 44RNA 445RNA 1RNA 1RNA 10RNA 10RNA 19RNA 19RNA 28RNA 28RNA 37RNA 37RNA 45RNA 456RNA 2RNA 2RNA 11RNA 11RNA 20RNA 20RNA 29RNA 29RNA 38RNA 38RNA 46RNA 467RNA 3RNA 3RNA 12RNA 12RNA 21RNA 21RNA 30RNA 30RNA 39RNA 39RNA 47RNA 478RNA 4RNA 4RNA 13RNA 13RNA 22RNA 22RNA 31RNA 31RNA 40RNA 40RNA 48RNA 489RNA 5RNA 5RNA 14RNA 14RNA 23RNA 23RNA 32RNA 32RNA 41RNA 41RNA 49RNA 49
ABCDEFGHIJKL1GI.1GI.1RNA 6RNA 6RNA 15RNA 15RNA 24RNA 24RNA 33RNA 33RNA 41RNA 412GI.2GI.2RNA 7RNA 7RNA 16RNA 16RNA 25RNA 25RNA 34RNA 34RNA 42RNA 423GI.1aGI.1aRNA 8RNA 8RNA 17RNA 17RNA 26RNA 26RNA 35RNA 35RNA 43RNA 434  NTC    NTC  RNA 9RNA 9RNA 18RNA 18RNA 27RNA 27RNA 36RNA 36RNA 44RNA 445RNA 1RNA 1RNA 10RNA 10RNA 19RNA 19RNA 28RNA 28RNA 37RNA 37RNA 45RNA 456RNA 2RNA 2RNA 11RNA 11RNA 20RNA 20RNA 29RNA 29RNA 38RNA 38RNA 46RNA 467RNA 3RNA 3RNA 12RNA 12RNA 21RNA 21RNA 30RNA 30RNA 39RNA 39RNA 47RNA 478RNA 4RNA 4RNA 13RNA 13RNA 22RNA 22RNA 31RNA 31RNA 40RNA 40RNA 48RNA 489RNA 5RNA 5RNA 14RNA 14RNA 23RNA 23RNA 32RNA 32RNA 41RNA 41RNA 49RNA 49
?. [Reaction set-up]
In a dedicated Mastermix hood, UV a 96 well qPCR plate and a Microseal B film for 30 minutes prior to use.Thaw reagents during this time. Prepare a 10 µM primer mix of GI.1 fwd; GI.1c_rev; GI.2 fwd; GI.2 rv; GI.1a fwd; GI.1a rv. Combine 10 µl of each primer stock (at 100 µM) with 40 µl of nuclease-free water.Prepare a Mastermix for number of required reactions + 7% extra to account for pipetting inaccuracies:    Distribute 14 µl of Mastermix to each well of 96 well PCR plate.Add 1 µl of template to respective wells (in duplicate).            - Ideally, this is done in a dedicated template addition areaSeal plate with Microseal B film. Briefly spin plate down. Place plate in qPCR thermocycler (BioRad CFX96) and commence run.   AB1µl per reaction2Nuclease-free H2O53SensiFAST Probe No-ROX One-Step Mix (2x)7.5410µM primer mix0.6510µM GI.1c probe0.15610µM GI.2 probe0.15710µM GI.1a probe0.158RiboSafe RNase Inhibitor0.159Reverse transcriptase0.310Template RNA111TOTAL REACTION VOLUME15 µl  ABC1NameSequence (5' - 3')Ref2GI.1 fwdTTCAGTTYTGGTAYGCCAATGCTG This study3GI.1c_revAGGCCTGCACAGTCGTAACGTTHall et al, 20184GI.1c probeFAM - ATGTCGTTCGTACCCTTTAACGGCCCTG - BHQ1This study5GI.2 fwdTGGAACTTGGCTTGAGTGTTGA Duarte et al, 20156GI.2 rvACGAGCGTGCTTGTGGACGG Duarte et al, 20157GI.2 probeHEX - TGTCAGAACTTGTTGAYATYCGCCC - BHQ1Duarte et al, 20158GI.1a fwdAGTGCAAGTTWTKCTGGGAACAACT This study9GI.1a rvAAGCCTGYACAGTCGTAGCAGC This study10GI.1a probeTexasRed - ATGTCATTCGTGCCYTTTAACAGCCCCA - BHQ2This studyDistribute 14 µl of Mastermix to each well of 96 well PCR plate.Add 1 µl of template to respective wells (in duplicate).            - Ideally, this is done in a dedicated template addition areaSeal plate with Microseal B film. Briefly spin plate down. Place plate in qPCR thermocycler (BioRad CFX96) and commence run.             - Ideally, this is done in a dedicated template addition areaSeal plate with Microseal B film. Briefly spin plate down. Place plate in qPCR thermocycler (BioRad CFX96) and commence run.   ABC1Temperature (°C)Duration2Reverse transcription4510 min3Initial denaturation952 min440 cycles of:955 sec56320 sec
AB1µl per reaction2Nuclease-free H2O53SensiFAST Probe No-ROX One-Step Mix (2x)7.5410µM primer mix0.6510µM GI.1c probe0.15610µM GI.2 probe0.15710µM GI.1a probe0.158RiboSafe RNase Inhibitor0.159Reverse transcriptase0.310Template RNA111TOTAL REACTION VOLUME15 µl
ABC1NameSequence (5' - 3')Ref2GI.1 fwdTTCAGTTYTGGTAYGCCAATGCTG This study3GI.1c_revAGGCCTGCACAGTCGTAACGTTHall et al, 20184GI.1c probeFAM - ATGTCGTTCGTACCCTTTAACGGCCCTG - BHQ1This study5GI.2 fwdTGGAACTTGGCTTGAGTGTTGA Duarte et al, 20156GI.2 rvACGAGCGTGCTTGTGGACGG Duarte et al, 20157GI.2 probeHEX - TGTCAGAACTTGTTGAYATYCGCCC - BHQ1Duarte et al, 20158GI.1a fwdAGTGCAAGTTWTKCTGGGAACAACT This study9GI.1a rvAAGCCTGYACAGTCGTAGCAGC This study10GI.1a probeTexasRed - ATGTCATTCGTGCCYTTTAACAGCCCCA - BHQ2This study
ABC1Temperature (°C)Duration2Reverse transcription4510 min3Initial denaturation952 min440 cycles of:955 sec56320 sec
?. [Analysis]
Set baseline to 100 RFU for each fluorophoreQuality control: No amplification in any channel in NTCs.FAM positive for GI.1 control only (or pooled positive if run). HEX positive for GI.2 control only (or pooled positive if run).TexasRed positive for GI.1a control only (or pooled positive if run).Cq of positive controls within 0.5 Cq of previous runs.Cq standard deviation