M13 primers

To generate a CPV-2 standard DNA for the real-time RPA, a PCR product containing 1755 bp covering the region of interest of VP2 was amplified from the CPV-2a DNA using VP2-Forward and VP2-Reverse as primers (Table 2) and cloned into the pMD19-T vector using the pMD19-T Vector Cloning Kit (Takara) according to manufacturer’s instructions. The resulting plasmid, pCPV-VP2, was transformed into Escherichia coli DH5α cells, and the positive clones were confirmed by sequencing using M13 primers (Invitrogen®, Carlsbad, CA, USA). pCPV-VP2 was purified with the SanPrep Plasmid MiniPrep Kit (Sangon Biotech Co., Ltd., Shanghai, China) and quantified using a ND-2000c spectrophotometer. The copy number of DNA molecules was calculated by the following formula: amount (copies/μL) = [DNA concentration (g/μL) / (plasmid length in base pairs × 660)] × 6.02 × 10²³.Table 2

Sequences of primers and probes for CPV-2 PCR, real-time PCR and real-time RPA assay

Name	Sequence 5′-3’Amplication size (bp)
VP2-FP	ATGAGTGATGGAGCAGTTCAACCAGAC	1775
VP2-RP	TTAATATAATTTTCTAGGTGCTAGTTGA
CPV-FP	AAACAGGAATTAACTATACTAATATATTTA	93
CPV-RP	AAATTTGACCATTTGGATAAACT
CPV-P	FAM-TGGTCCTTTAACTGCATTAAATAATG TACC-BHQ1
CPV-RPA-FP	CACTTACTAAGAACAGGTGATGAATTTGCT ACAG	214
CPV-RPA-RP	AGTTTGTATTTCCCATTTGAGTTACACCACGTCT
CPV-RPA-P	CCTCAAGCTGAAGGAGGTACTAACTTTGGT/BHQ1-dT//THF //FAM-dT/ATAGGAGTTCAACAAG-C3 spacer

Aimed to generate a M. bovis-standard DNA for the RPA assays, a PCR product with 1,908 bp covering the region of interest of uvrC gene, was amplified from the M. bovis DNA using uvrC-F and uvrC-R as primers (Table 1) and cloned into the pMD19-T (Takara, Dalian, China) for standards. The generating plasmid, pMbovis-uvrC, was transformed into Escherichia coli DH5α cells and the positive clones were identified by sequencing with M13 primers (Invitrogen^®, Carlsbad, CA, USA). pMbovis-uvrC was purified with the SanPrep Plasmid MiniPrep Kit (Sangon Biotech, Shanghai, China) and quantified with a ND-2000c spectrophotometer. The copy number of DNA molecules was calculated according to the formula as follows: amount (copies/μl) = [DNA concentration (g/μl)/(plasmid length in base pairs × 660)] × 6.02 × 10²³. Aliquots of the standard DNA were prepared in 10-fold serial dilutions from 1.0 × 10⁷ to 1.0 × 10⁰ copies/μl in nuclease-free water and stored at −80°C until use.

A polymerase chain reaction (PCR) was performed using the primers, psbA-F (5′-GTN GAY ATH GAY GGN ATH MGN GAR CC-3′) and psbA-R (5′-GGR AAR TTR TGN GCR TTN CKY TCR TGC-AT-3′) [42] (link). The PCR reaction mixture (50 µL) consisted of 25 µL ExTaq premix (TaKaRa, Dalian, China), 0.5 µM each primer, and 2 µL (ca. 10 ng DNA) of template. The amplification conditions comprised steps at 95°C for 5 min, 30 cycles at 94°C for 1 min, 55°C for 1 min, and 68°C for 1 min followed by one step of 10 min at 72°C. The amplified products were gel-purified and ligated into the pMD18-T vector (TaKaRa, Dalian, China) and then transformed into competent cells of Escherichia coli DH5a. The ampicillin-resistant clones were randomly picked and screened for inserts using performing colony PCR with M13 primers (Invitrogen, Shanghai, China) for the vector.