Sequencing of Canine Parvovirus (CPV) from Rectal Swab Samples V.1

Sara França de Araújo dos Santos, Ueric José Borges de Souza, Martha Trindade Oliveira, Jairo Jaime, Fernando Rosado Spilki, Ana Cláudia Franco, Paulo Michel Roehe, Fabrício Souza Campos

Published: 2023-07-14 DOI: 10.17504/protocols.io.36wgq3563lk5/v1

Disclaimer

Abstract

Canine parvovirus (CPV) is a highly contagious viral disease that affects dogs, especially puppies. CPV-2 is recognized for its resilience in contaminated environments, ease of transmission among dogs, and pathogenicity for puppies. In this protocol, we have adapted the methodology to allow the recovery of complete CPV-2 genomes directly from clinical samples (dry swabs) from puppies with clinical signals of viral enteritis. A multiplex PCR was designed with primers targeting fragments of 400 to 1,000 base pairs (bp) along the full length of the viral genome. The resulting reads were compared after sequencing with the Nanopore technology. Genome assembly revealed that the smaller fragments generated larger numbers of reads, allowing a more reliable coverage of the genome than those attained with primers targeting larger amplicons. Both new methodologies were efficient in amplification and sequencing.

Steps

Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)

This protocol uses for extraction.

All kit's solutions need to be prepared beforehand, as described by the kit. If you've already prepared the solution, go to step 2. This is a modified version of the kit's protocol. The original kit's protocol can be found here.

1.1.

DNA/RNA Buffer:

Add 500µL of beta-mercaptoehtanol (user supplied) per 100 ml Viral DNA/RNA Buffer , (final 0.5% (v/v)

1.2.

DNA/RNA Wash 1:

Kit R2140 --> Add 20mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 1 concentrate

Kit R2141 --> Add 80mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 1 concentrate

1.3.

DNA/RNA Wash 2:

Kit R2140 --> Add 30mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 2 concentrate

Kit R2141 --> Add 120mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 2 concentrate

1.4.

DNA/RNA Shield™:

Kit R2140 --> Add 25mL of nuclease-free water (user supplied) to 2X DNA/RNA Shield™ concentrate

Kit R2141 --> Add 125mL of nuclease-free water (user supplied) to 2X DNA/RNA Shield™ concentrate

1.5.

Proteinase K (20 mg/ml):

Kit R2140 --> Add 1.04mL of Proteinase K Storage Buffer to lyophilized Proteinase K (20mg)

Kit R2141 --> Add 3.12mL of Proteinase K Storage Buffer to lyophilized Proteinase K (60mg)

Mix by vortexing. Use immediately or store frozen aliquots.

Elute each dry Sample into 400µL of 1X viral DNA/RNA buffer.

2.1.

Our samples were dry rectal swabs of animals confirmed positive by a rapid test.

Samples were collected and stored in a centrifuge tube (without transporting media) at -20 ºC until processing.

For each sample, add 10µL of beads and 4µL of proteinase K.

Obs: when working with many samples, one can always prepare a mixed solution with both components. Just make to keep the beads suspended while pipetting.

3.1.

Transfer the plate (or your tubes) to magnetic stand (user supplied). After the beads have pelleted, aspirate and discard the supernatant.

This protocol can be fast tracked by the use of an automated extractor .

We use EXTRACTA 96 (Loccus).

Perform one wash of the pellet (beads) with 250µL of MagBead DNA/RNA Wash 1 solution.

Perform one wash of the pellet (beads) with 250µL of MagBead DNA/RNA Wash 2 solution.

Add 250µL of 80% ethanol and mix well.

Transfer the sample (liquid and beads) to a new place/tube.

Pellet the beads and discard the the supernatant.

Add 50µL of nuclease-free water and mix well (this is the elution step).

Pellet the beads (magnetically) and transfer the supernatant (containing DNA/RNA) to a new plate/tube.

Multiplex PCR to obtain CPV genome fragments

10.

This protocol uses 2 sets of oligos: one set amplifies ~400 bp sequences that overlap about 100 nucleotides with each other (Table 1); the other amplifies ~1000 bp sequences that overlap about 100 nucleotides with each other (Table 2). Primers are listed in the tables below and were design based on Canine parvovirus reference sequence NC_001539.1 (GenBank).

Primers should be mix into 2 pools for each set. To prepare a pool, add 5µL of each primer (at 100micromolar (µM)) as indicated. This will be your 10X (stock) solution. Working solution should be dilute to 1X concentration (10micromolar (µM)).

A	B	C	D	E	F
CPV-400_0.7_LEFT*	1	ATGTCTGGCAACCAGTATACTG	22	45.50	60.30
CPV-400_1_LEFT	1	AACCAACTGACCAAGTTCACGT	22	45.45	60.80
CPV-400_1_RIGHT	1	GTTCCAGCGAACATCCTTTCCA	22	50.00	61.31
CPV-400_1.5_LEFT*	1	AGGTGGCGGGCTAATTGTG	19	57.90	59.50
CPV-400_2_LEFT	2	AAGAAACATGCAGAAAATGAAGCATT	26	30.77	59.73
CPV-400_2_RIGHT	2	CGTAGCCATTTACCAGTTGCTTG	23	47.83	60.67
CPV-400_3_LEFT	1	ATGGGGAAAAGATCAAGGCTGG	22	50.00	60.81
CPV-400_3_RIGHT	1	AGTGTGCTGACAATTTGTCTGTC	23	43.48	59.94
CPV-400_4_LEFT	2	GGGTGACTATATTAACATACAGACATAAGC	30	36.67	60.59
CPV-400_4_RIGHT	2	TCCTGGTTGTGCCATCATTTCA	22	45.45	60.68
CPV-400_5_LEFT	1	ACTTTGCGGGACTTGGTTAGTA	22	45.45	59.81
CPV-400_5_RIGHT	1	ACAACCAACATTACCCACAGCT	22	45.45	60.61
CPV-400_6_LEFT	2	CAGTTCTTTTTCATGGACCAGCA	23	43.48	59.93
CPV-400_6_RIGHT	2	AAACCAAAGTCTCCTGGAAGCT	22	45.45	60.01
CPV-400_7_LEFT	1	TGGATGTGAAGAAAGACCTGAACA	24	41.67	60.47
CPV-400_7_RIGHT	1	AACGCCAAGTTGGTTTGATTGT	22	40.91	60.01
CPV-400_8_LEFT	2	AGTGGACCTTGCACTGGAAC	20	55.00	60.20
CPV-400_8_RIGHT	2	GCTTCGTCGTGTTCTTTTGCAG	22	50.00	61.33
CPV-400_9_LEFT	1	AAATATCTTGGGCCTGGGAACA	22	45.45	59.87
CPV-400_9_RIGHT	1	ACTGCTCCATCACTCATTGGTG	22	50.00	60.80
CPV-400_10_LEFT	2	ACCACCTCATATTTTCATCAATCTTGC	27	37.04	60.91
CPV-400_10_RIGHT	2	TCAACCAATGACCAAGGTGTTACA	24	41.67	60.95
CPV-400_11_LEFT	1	GTGGTTGTAAATAATATGGATAAAACTGCA	30	30.00	60.00
CPV-400_11_RIGHT	1	TGTTCTATCCCATTGAAAATAATATCTCCA	30	30.00	59.80
CPV-400_12_LEFT	2	TGCCATTTACTCCAGCAGCTAT	22	45.45	60.01
CPV-400_12_RIGHT	2	TCCCATTTGAGTTACACCACGT	22	45.45	60.08
CPV-400_13_LEFT	1	TTTGCCTCAATCTGAAGGAGCT	22	45.45	60.14
CPV-400_13_RIGHT	1	ATCATTCGTTACAGGAAGGTTAAAGTT	27	33.33	59.83
CPV-400_14_LEFT	2	ACACCTGAGAGATTTACATATATAGCACA	29	34.48	60.63
CPV-400_14_RIGHT	2	ACCTTTCCACCAAAAATCTGAGTAAG	26	38.46	60.18
CPV-400_15_LEFT	1	CAAATGGTCAAATTTGGGATAAAGAATTTG	30	30.00	60.15
CPV-400_15_RIGHT	1	TTCTAGGTGCTAGTTGATATGTAATAAACA	30	30.00	59.56
CPV-400_16_LEFT	2	TGTTTATTACATATCAACTAGCACCTAGAA	30	30.00	59.56
CPV-400_16_RIGHT	2	TCTAAGGGCAAACCAACCAACC	22	50.00	61.20
CPV-400_17_LEFT	1	AGGTTTGTTAGATGGTATACAATAACTGT	29	31.03	59.61
CPV-400_17_RIGHT	1	AGCTTTAAATACTAATTTACCTTTCCACCA	30	30.00	60.50
CPV-400_17.5_RIGHT*	1	AAGTATCAATCTGTCTTTAAGGGG	24	37.50	60.10
CPV-400_18_LEFT*	2	TATAAGGTGAACTAACCTTACCATA	25	32.00	59.20
CPV-400_18_RIGHT*	2	TTAATATAATTTTCTAGGTGCTAGTTG	27	25.90	59.20

Table 1. Primers targeting 400 bp amplicons

A	B	C	D	E	F
CPV-1000_1_LEFT	1	CTGACCAAGTTCACGTACGTATGA	24	45.83	60.93
CPV-1000_1_RIGHT	1	TGTTCAGTGTAAAGTGTGCTGACA	24	41.67	61.18
CPV-1000_2_LEFT	2	GTGAATGGGTGACTATATTAACATACAGAC	30	36.67	60.73
CPV-1000_2_RIGHT	2	ACCAAACCAAAGTCTCCTGGAAG	23	47.83	60.95
CPV-1000_3_LEFT	1	AAGCAAATTGAACCAACTCCAGT	23	39.13	59.55
CPV-1000_3_RIGHT	1	GGTGGTGGTTTACTTCTTTTAGTTGG	26	42.31	60.90
CPV-1000_4_LEFT	2	CTAAGGACGCTAAAGATTGGGGG	23	52.17	61.00
CPV-1000_4_RIGHT	2	GTTCCTGTAGCAAATTCATCACCTG	25	44.00	60.77
CPV-1000_5_LEFT	1	CCATCTCATACTGGAACTAGTGGC	24	50.00	60.82
CPV-1000_5_RIGHT*	1	TGGATTCCAAGTATGAGAGGCTCT	24	45.83	61.21
CPV-1000_6_LEFT	2	AACCAAGACTTCATGTAAATGCACC	25	40.00	60.66
CPV-1000_6_RIGHT*	2	TGGATTCCAAGTATGAGAGGCTCT	24	45.83	61.21

Table 2. Primers targeting 1000 bp amplicons

11.

We used to perform PCR.

Reactions should be set up independent for each pool , with a final volume of 25µL, as bellow:

Reactions for 400 bp products:

    * Master Mix 2X         - `12.5µL`



    * Primer pool 1  **or**  2   -   `1.6µL`  



    * Nuclease-free H20 -  `5.9µL`



    * DNA                         -   `5.0µL`

Reactions for 1000 bp products:

    * Master Mix 2X         - `12.5µL`



    * Primer pool 1  **or**  2   -  `0.4µL`  



    * Nuclease-free H20 -  `7.1µL`



    * DNA                         -  `5.0µL`

11.1.

The final concentration in a reaction should be 15nanomolar (nM) per primer. Therefore, the volume of primers used in a reaction will vary according with the number of primers in the pool. More about this subject can be found in Quick et al. (2017).

12.

PCR's run method should be set as:

    * `98°C` for `0h 3m 0s`



    * 35 cycles of (`98°C` for `0h 0m 15s`; `63°C` for `0h 5m 0s`)



    * Hold at `4°C`

Purifying PCR Products with AMPure XP beads (Beckman Coulter)

13.

Mix amplification products of Pool 1 and Pool 2 (final volume 50µL).

14.

We purify the fragments with , using a modified protocol (original protocol ca be found here).

14.1.

This is step can also be fast-tracked by the use of an automated system.

15.

Add an equal volume of AMPure XP per sample (50µL).

16.

After biding, on a magnetic stand, wash beads once with 80% (v/v) ethanol.

17.

Elute DNA with 20µL of (sequencing appropriate) buffer.

18.

Transfer DNA to a new plate/tube.

19.

Quantify your DNA.

19.1.

We used

Equipment

Value	Label
Qubit™ 3 Fluorometer	NAME
Fluorometer for nucleic acid quantitation	TYPE
Invitrogen	BRAND
Q33216	SKU
Fluorometer for nucleic acid quantitation	SPECIFICATIONS

Library preparation & sequencing

20.

To prepare a library, use the Ligation and Barcoding kits (following manufacturer's instructions).

We used Ligation Sequencing kit SQK-LSK-109 and Native Barcoding kits EXP-NBD104 and EXP-NBD114 (Oxford Nanopore)

20.

21.

Load library on your flow cell and sequence your sample.

21.1.

We used R9.4 Oxford MinION flow cell (FLO-MIN106) and MinION Mk1B device for sequencing.