Rabbit calicivirus capsid Taqman RT-qPCR

A	B	C	D	E	F	G	H	I	J	K	L
GI.1	GI.1	RNA 6	RNA 6	RNA 15	RNA 15	RNA 24	RNA 24	RNA 33	RNA 33	RNA 41	RNA 41
GI.2	GI.2	RNA 7	RNA 7	RNA 16	RNA 16	RNA 25	RNA 25	RNA 34	RNA 34	RNA 42	RNA 42
GI.1a	GI.1a	RNA 8	RNA 8	RNA 17	RNA 17	RNA 26	RNA 26	RNA 35	RNA 35	RNA 43	RNA 43
NTC	NTC	RNA 9	RNA 9	RNA 18	RNA 18	RNA 27	RNA 27	RNA 36	RNA 36	RNA 44	RNA 44
RNA 1	RNA 1	RNA 10	RNA 10	RNA 19	RNA 19	RNA 28	RNA 28	RNA 37	RNA 37	RNA 45	RNA 45
RNA 2	RNA 2	RNA 11	RNA 11	RNA 20	RNA 20	RNA 29	RNA 29	RNA 38	RNA 38	RNA 46	RNA 46
RNA 3	RNA 3	RNA 12	RNA 12	RNA 21	RNA 21	RNA 30	RNA 30	RNA 39	RNA 39	RNA 47	RNA 47
RNA 4	RNA 4	RNA 13	RNA 13	RNA 22	RNA 22	RNA 31	RNA 31	RNA 40	RNA 40	RNA 48	RNA 48
RNA 5	RNA 5	RNA 14	RNA 14	RNA 23	RNA 23	RNA 32	RNA 32	RNA 41	RNA 41	RNA 49	RNA 49

Plate layout for rabbit calicivirus capsid Taqman RT-qPCR. Samples and controls are routinely run in duplicate. We run individual controls for each strain; at a minimum a pooled positive control should be run.

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:

A	B
	µl per reaction
Nuclease-free H2O	5
SensiFAST Probe No-ROX One-Step Mix (2x)	7.5
10µM primer mix	0.6
10µM GI.1c probe	0.15
10µM GI.2 probe	0.15
10µM GI.1a probe	0.15
RiboSafe RNase Inhibitor	0.15
Reverse transcriptase	0.3
Template RNA	1
TOTAL REACTION VOLUME	15 µl

Preparation of Mastermix. Include 7% extra to account for pipetting inaccuracies.

A	B	C
Name	Sequence (5' - 3')	Ref
GI.1 fwd	TTCAGTTYTGGTAYGCCAATGCTG	This study
GI.1c_rev	AGGCCTGCACAGTCGTAACGTT	Hall et al, 2018
GI.1c probe	FAM - ATGTCGTTCGTACCCTTTAACGGCCCTG - BHQ1	This study
GI.2 fwd	TGGAACTTGGCTTGAGTGTTGA	Duarte et al, 2015
GI.2 rv	ACGAGCGTGCTTGTGGACGG	Duarte et al, 2015
GI.2 probe	HEX - TGTCAGAACTTGTTGAYATYCGCCC - BHQ1	Duarte et al, 2015
GI.1a fwd	AGTGCAAGTTWTKCTGGGAACAACT	This study
GI.1a rv	AAGCCTGYACAGTCGTAGCAGC	This study
GI.1a probe	TexasRed - ATGTCATTCGTGCCYTTTAACAGCCCCA - BHQ2	This study

Primer and probe sets for rabbit calicivirus capsid TaqMan RT-qPCR

• 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 area

• Seal plate with Microseal B film.

• Briefly spin plate down.

• Place plate in qPCR thermocycler (BioRad CFX96) and commence run.

A	B	C
	Temperature (°C)	Duration
Reverse transcription	45	10 min
Initial denaturation	95	2 min
40 cycles of:	95	5 sec
	63	20 sec

Thermocycling conditions for rabbit calicivirus capsid TaqMan RT-qPCR

Set baseline to 100 RFU for each fluorophore* Quality control:

1. No amplification in any channel in NTCs.
2. FAM positive for GI.1 control only (or pooled positive if run).
3. HEX positive for GI.2 control only (or pooled positive if run).
4. TexasRed positive for GI.1a control only (or pooled positive if run).
5. Cq of positive controls within 0.5 Cq of previous runs.
6. Cq standard deviation <0.5 between replicates.