Pcr clean up system kit

Fifteen micrograms of RNA was used to determine the cDNA 5′-end (Bensing et al., 1996 (link); Saito et al., 2009 (link)). RNAs were prepared either with or without RNA 5′-Pyrophosphohydrolase (RppH) (New England BioLabs) to distinguish primary transcript 5′-ends from internal 5′-processing sites. DNA primers Jrev and Erev were used for cDNA synthesis with SuperScript III Reverse Transcriptase (Invitrogen) after fusing the GeneRacer RNA Oligo to the isolated RNA. Additional primers for subsequent PCR amplification of cDNAs were GeneRacer 5′-nested primer, homologous to the adaptor GeneRacer RNA oligo, Erev2 and Jrev2. PCR products that were detected both with and without tobacco acid pyrophosphatase treatment were purified by using a PCR clean-up system kit (Promega) and cloned by using the pGEM-T Easy kit (Promega), and three clones of each candidate were sequenced.

The final PCR products were observed via electrophoresis on a 1.4% agarose gel at 100 V for 30 min. When the multi-band was formed, gel purification was performed, and when the single band was formed, PCR purification was performed. PCR and gel purification were conducted via Wizard® SV gel and PCR Clean-up System Kit (Promega, San Luis Obispo, CA, USA) according to the manufacturer’s instructions. The purified PCR product was sequenced in both directions via Bionics Co., Ltd. (Seoul, Korea) using EF1 and EF2 primer for TEF and 5f2, 7cr, 7cF, and 11aR primers for RPB2 (Supplementary Table S7). The consensus sequences were assembled and revised using the Seqman program (DNASTAR, Madison, USA)^{37 (link)}. The novel sequences generated in this study were deposited in National Centre for Biotechnology Information (NCBI) GenBank.

DNA was extracted according to the manufacturer’s instructions using the AllPrep DNA/RNA minikit (Qiagen, United States). The hypervariable region V3-V4 of the protistan 18S rRNA and the bacterial 16S rRNA genes were amplified using eukaryote-specific primers (F1, 5′-CCA GCA SCY GCG GTA ATT CC-3′; R3, 5′-ACT TTC GTT CTT GAT YRA-3′) (62 (link)) and bacterium-specific primers (341F, 5′-CCT AYG GGR BGC; 806R, 5′-GGA CTA CNN GGG TAT CTA AT-3′) (63 (link)). PCR conditions followed those described in references 62 (link) and 63 (link). It should be noted that haptophytes, a significant assemblage of protists, may be underestimated when these universal primer sets are used, due to the fact that a single base mismatch on the 3′ end of the reverse primers and their GC-rich genomes may impede amplification (64 (link)). Each sample was amplified in triplicate, pooled, and purified using the Wizard SV gel and PCR clean-up system kit (Promega, USA). All purified fragments were sequenced on the Illumina MiSeq PE300 sequencing platform by MajorBio Bioinformatics Technology Co., Ltd. (Shanghai, China).

To confirm the presence of important antimicrobial resistance genes (bla_OXA-51, bla_OXA-23, and mcr-1) in isolates positive for these genes, the amplified products were purified using the commercial kits: Wizard^® SV Gel and PCR Clean-Up System Kit (Promega Corporation, Madison, USA). In addition, to prove that these three isolates were indeed Pseudomonas aeruginosa, we amplified and sequenced the 16S region of these isolates using bacterial Universal primers: 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) following the protocol previously described [29 ]. The sequencing of purified PCR products was performed with ABI Prism BigDye Kit on the ABI 3130 Genetic Analyzer (Applied Biosystems). The samples were sequenced at least three times in each direction of the tape, making a total of six sequences from the same sample. All sequencing assays were done by Myleus Biotecnologia, based in Belo Horizonte, MG. The sequence electropherograms were analyzed with ChromasPro software (http://www.technelysium.com.au/chromas.html (accessed on 23 May 2020)). The similarity between the sequences was verified with BLASTn tool (Basic Alignment Search Tool–(http://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 23 May 2020).

The ITS1-rDNA was amplified by conventional PCR using the primers L5.8S: 5′-TGATACCACTTATCGCACTT-3′ and LITSR: 5′-CTGGATCATTTTCCGATG-3′ [14 (link)]. Amplification reactions were performed in volumes of 50 μL. Amplicons from the PCR-positive samples (300–350 bp, depending on the species) were visualized on a 2% agarose gel and purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega, Madison, USA). The products were then sequenced with the same primers used in the PCR assay. Sequencing was performed on an automated sequencer at Plataforma de Sequenciamento Genômico ABI-3730 (Oswaldo Cruz Institute/FIOCRUZ).
Sequence alignment was performed using SeqMan Pro (DNASTAR) and comparisons were conducted with Leishmania reference strains sequences obtained from the GenBank database. Phylogenetics analyses with the evolutionary history was inferred using the maximum likelihood method based on the Jukes–Cantor model, and the sequences were aligned using Molecular Evolutionary Genetic Analysis (MEGA) version 6. This same software was used to calculate a distance matrix and the genetic distance percentage between the test samples and reference strains of Leishmania spp.

Sanger sequencing of the internal transcribed spacer of ribosomal DNA (ITS1-rDNA) was performed only in one case where it was not possible to obtain taxonomic identification through MLEE or PCR-RFLP.
The ITS1-rDNA was amplified by conventional PCR using the primers L5.8S: 50-TGATACCACTTATCGCACTT-30 and LITSR: 50-CTGGATCATTTTCCGATG-30. Amplification reaction was performed in volume of 50 μL. Amplicons from the PCR positive sample were visualized on 2% agarose gel and purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega, Madison, Wisconsin, USA). The products were then sequenced with the same primers used in the PCR assay. Sequencing was performed on an automated sequencer at Plataforma de Sequenciamento Genômico ABI-3730 (Oswaldo Cruz Institute/Fiocruz).
Sequence alignment was performed using SeqMan Pro (DNASTAR, Madison, Wisconsin, USA) and comparisons were conducted with Leishmania reference strains sequences obtained from the GenBank database. Phylogenetics analyses with the evolutionary history were inferred using the maximum likelihood method based on the Jukes-Cantor model and the sequences were aligned using Molecular Evolutionary Genetic Analysis (MEGA) version 6 (Tokyo Metropolitan University, Tokyo, Japan; Arizona State University, Arizona, USA; King Abdulaziz University, Jeddah, Saudi Arabia).

To confirm the identity and further characterize the LSDV positive samples, four genes: the RPO30, GPCR, Extracellular enveloped virus (EEV) glycoprotein, and CaPV homolog of the variola virus B22R genes were amplified by PCR as previously described using the primers listed in Table 2. All PCRs were performed in a 20 μL reaction volume containing 500 nM of each of the forward and the reverse primers, 200 uM of dNTPs, 1x PCR buffer (Qiagen), 1.25 U of Taq DNA polymerase (Qiagen), and 2 μL template DNA. After amplification, the PCR products were analyzed on a 2% gel electrophoresis and visualized on a Gel Documentation System (Bio-Rad). The PCR products were purified using the PCR clean-up system kit (Promega), following the manufacturer’s instructions. The products were sequenced using both forward and reverse primers at LGC Genomics (Germany). The nucleotide sequences were deposited in the GenBank database under the accession numbers OL689584 to OL689619.