Pmd18 t vector cloning kit

A total of 113 fecal samples (30 CPV-2 fecal samples of known genotypes collected in 2013, and 83 field samples collected in 2017 and 2019), four batches of CPV-2 vaccines, and six plasmids (two as reference and four as control plasmids) were used in this study (Supplementary Table S1). The plasmid p-CPV-2 was synthesized based on the VP2 gene of CPV-b (original CPV-2; GenBank accession no. M38245). Five plasmids of pCPV-2a, pCPV-2b, pCPV-2c, pCPVpf, and pCPVint were generated using the pMD18-T Vector Cloning Kit (Takara, Dalian, China), and the insert fragments of the plasmids were amplified from CPV-js1 (CPV-2a; GenBank accession no. KJ754512), CPV-js2 (CPV-2b; KJ754513), CPV-js4 (CPV-2c; KJ754515), CPVpf (Yoon et al., 2009 (link)) (CPVpf; isolated from commercial Vanguard^® Plus 5, Pfizer Inc., Lincoln, NE, United States), and CPVint (Yoon et al., 2009 (link)) (CPVint; isolated from commercial vaccine Nobivac^® DHPPi, Intervet Inc., Netherlands) using primer set CPV-1270F/1270R (Table 1), respectively.
Viral RNA/DNA was extracted using the MiniBEST Viral RNA/DNA Extraction Kit version 4.0 (Takara, Dalian, China) according to the manufacturer’s instructions.

Sixteen clone libraries of actinobacterial 16S rRNA genes were constructed using the pMD18-T Vector Cloning Kit and E. coli DH5α competent cells (Takara, Dalian) following the manufacturer′s instructions. The positive clones from each library inoculated on MacConkey agar with ampicillin (100 μg/ml) were randomly picked and sequenced using M13F (−47) primer on ABI 3730xl capillary sequencers (Applied Biosystems, USA).

For the sequencing protocol, the PCR product of some positive samples was chosen. All positive samples for each pathogen were sequenced if their numbers were fewer than ten. However, if their numbers were more than ten, we sequenced at least 30% of the positive samples from different study areas. These samples represented different livestock species from various sampling areas included in this study. The PCR product of the positive samples was purified using the EasyPure Quick Gel Extraction Kit (TransGen Biotech, Beijing, China) and cloned into E. coli DH5α cells (Takara, Shiga, Japan) using the pMD™18-T Vector Cloning Kit (Takara, Shiga, Japan). Plasmid purification was performed using the EasyPure^® Plasmid MiniPrep Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s manual. Subsequently, at least three positive clones were sent to Genewiz company, Suzhou, China, for sequencing.
The good-quality sequence reads obtained in this study were compared with published sequences deposited in the GenBank databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 7 October 2022)) using the BLASTn search tool. Phylogenetic trees were constructed using the maximum likelihood method in MEGA X with the bootstrap method, employing 1000 replications [32 (link)].

Adult males were sampled daily from day 1 to 30 after emergence (DAE). Total RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Total RNA was incubated with 10 U DNase I (Thermo Scientific, USA) at 37 °C for 30 min for mRNA purification. First strand cDNA was produced from 5 μg RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Target gene sequences (lola, topi, per, aly, rac, rho, upd, magu) were obtained by retrieving previously constructed B. dorsalis transcriptome data (Y.-C. D., Z.-J. W., C.-Y. N., unpublished data), and their gene specific primers were designed using Primer Premier 5.0 (Premier, Canada) (Table S1). PCR amplicons were purified using AxyPrep DNA Gel Extraction Kit (AxyPrep, USA). The purified products were ligated to cloning vector by using pMD™ 18-T Vector Cloning Kit (TaKaRa, China). The plasmid recombinants (pMD-18T-lola, -topi, -per, -aly, -rac, -rho, -upd, -magu) were amplified by PCR and verified by Sanger sequencing (Invitrogen, Shanghai, China).

The standards of the ASPCR assays for A2063G and A2064G were then constructed. Plasmids containing a wild-type fragment of M. pneumoniae 23S rRNA full-length sequence were obtained by cloning the PCR products amplified by the primers 23SrRNA-F and 23SrRNA-R into pMD18-T (pMD™18-T Vector Cloning Kit, TaKaRa, Japan) vectors. The mutations of A2063G and A2064G were introduced into wild-type plasmids by sit-directed mutagenesis (TaKaRa MutanBEST Kit, TaKaRa, Japan) using primers 23SA2063G-F, 23SA2064G-F, and 23S2063/2064-R. The plasmids containing mutations were confirmed by sequencing and quantified by spectrophotometer. Serial 10-folds dilutions of plasmids ranging from 10 to 10⁶ copies/μL were made as standards for ASPCR.
The mutant-specific and nonspecific standard curves of A2063G and A2064G were obtained by amplifying the mutant standards with the corresponding primer sets. The ASPCR mix contained 12.5 μL power SYBR Green PCR Master 2 × Mix, 2 μL mutant-specific/non-specific upstream primer, 2 μL corresponding downstream-primer, 2 μL DNA templates, 6.5 μL ddH₂O. The reaction conditions were 50 °C for 2 min, followed by a denaturation step at 95 °C for 10 min, 40 cycles of real-time PCR amplification (95 °C for 15 s, 60 °C for 1 min) with a final dissociation stage (95 °C for 15 s, 60 °C for 30 s, 95 °C for 15 s). Each reaction was conducted in triplicate.