Easypure bacteria genomic dna kit

The whole genomic DNA of strain D-2 was extracted using EasyPure^® Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China), and the concentration and purity of DNA were detected using a NanoDrop-2000 spectrophotometer. The sequencing of the complete genome was executed by LC-BIO, Technology Co. Ltd (Hangzhou, China) using a hybrid of the HiSeq 2500 (Illumina, United States) and PacBio RS II (Pacific Biosciences, United States) platforms. The coding genes were annotated with the National Center for Biotechnology Information (NCBI) NR database by BLAST. The functions of coding genes were then annotated by the Gene Ontology (GO) database, and the pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The proteins encoded by genes were classified and annotated on databases of Clusters of Orthologous Groups (COG/KOG) and Carbohydrate-Active enzyme (CAZy).

Bacteria were grown in LB medium until OD₆₀₀ of 1.0, and genomic DNAs were extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA gene sequences were amplified using the primers 27f and 1492r (Coenye et al., 1999 (link)). The products were examined using 1% agarose gel electrophoresis and sent to Sangon Biotech Company in Shanghai, China, for sequencing. SeqMan V.5.00 was used to assemble sequences generated from forward and reverse primers.

Bacterial genomic DNA [i.e., Escherichia coli (E. coli.), Pasteurella multocida (P.M.), Rimerella anatipstifer (R.A.) and Salmonella spp. (S.S.)] were extracted using EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China).
Viral DNA [i.e., cGPV, N-GPV, MDPV, novel recombinant Muscovy duck parvovirus (N-MDPV), Duck adenovirus A (DAdV-A), duck enteritis virus (DEV), duck origin-goose haemorrhagic polyomavirus (GHPV)] and viral RNA [i.e., duck hepatitis virus type 1 and 3 (DHAV-1 and DHAV-3), Avian Tembusu virus (ATmV), H9N2 subtype avian influenza virus (AIV), Muscovy duck reovirus (MDRV) and novel duck reovirus (N-DRV)] were extracted using EasyPure Viral DNA/RNA Kit (TransGen Biotech, Beijing, China).
The cDNA of RNA viruses (DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV) was prepared with isolated RNA (approximately 100 ng for each) using TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).
DNA and cDNA were quantified using a NANODROP 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and stored at − 80 °C until use.

The pectinolytic isolates were first identified using 16S rDNA sequencing. The bacterial gDNA was isolated using an Easy-Pure Bacteria Genomic DNA Kit (TransGen Biotech) according to the protocol provided by the manufacturer. The 16S rDNA region was amplified by conventional PCR using the universal primers 27F and 1492R (Weisburg et al., 1991 (link)). The PCR reaction was performed using Taq Plus Master Mix (Vazyme Biotech). The PCR program was 94°C for 5 min, 30 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 50 s, and 72°C for 10 min. The PCR products were purified using a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech). The purified PCR amplicons were sequenced in the forward and reverse directions (Sangon Biotech). The sequenced reads were assembled into consensus sequences with primer trimming using Contig Express from the Vector NTI Suite v6.0. The 16S rDNA sequence for each isolate was searched against the NCBI 16S rRNA database using BLASTn.

Strain FH-1 was identified using the comparative analysis of 16S rRNA gene sequences. An EasyPure® Bacteria Genomic DNA Kit (TransGen Biotech Co., Ltd., Beijing, China) was used to extract the total genomic DNA of strain FH-1. PCR was applied to amplify the 16S rRNA gene using the universal forward and reserve primers of 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), respectively. Each PCR reaction contained Premix Taq 25 μl, DNA template 500 ng, 1492R, and 27F primers 10 μl, respectively, complemented by sterile water to a final volume of 50 μl. The PCR cycle was set up as the following steps: (1) preheating (95°C for 5 min), (2) 35 cycles of denaturation (95°C for 30 s), annealing (56°C for 30 s), and extension (72°C for 1.5 min), and (3) a final extension (10 min at 72°C). An EasyPure Quick Gel Extraction Kit (TransGen Biotech Co., Ltd., Beijing, China) was used to purify the amplified PCR products with the 1% agarose gel electrophoresis. The purified PCR products were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Phylogenetic and molecular evolutionary analyses were carried out using MEGA version 7.0 [21 (link)]. Phylogenetic trees were reconstructed using the neighbor-joining method [22 (link)] with bootstrap analysis of 1000 replicates.

The genomic DNA of the isolates were prepared using the EasyPure Bacteria Genomic DNA kit (TransGen Biotech, Beijing, China) and were screened for the presence of twelve virulence genes including adhesion related proteins (ptfA, tadD and pfhA), dermonecrotoxin (toxA), iron binding proteins (fur, tbpA and hgbB), sialidases (nanB), hyaluronidase (pmHAS) and outer membrane proteins (ompA, ompH and oma87). Primers used for ptfA, tadD, pfhA, toxA, fur, nanB, pmHAS, ompA, ompH and oma87 were described by Tang et al. [26 (link)]. Primers used for tbpA and hgbB were described by Ewers et al. [25 (link)]. The expected PCR products were purified and sequenced, and the identities of these products were confirmed by comparing the sequences against the NCBI GenBank database.

Viral RNAs (RNA viruses, i.e. AIV, ATmV, DHAV-1 and DHAV-3, MDRV) were extracted using EasyPure Viral DNA/RNA Kit (Tiangen, Beijing, China), viral DNAs (DNA viruses, i.e. DAdV-A, DEV, GPV and N-GPV) were extracted using EasyPure Micro Genomic DNA Kit (Transgen Bioteck, Beijing, China), according to instructions provided by the manufacturer. cDNAs (AIV, ATmV, DHAV-1 and DHAV-3, MDRV) were prepared with viral RNAs (with approximate 100 ng of viral RNA) using TransScript II All-in-One First-Strand cDNA Synthesis SuperMix (One-Step gDNA Removal) (Tiangen, Beijing, China). Bacteria genomic DNAs (bacteria pathogens, i.e. E. coli., P.M., R.A. and S.S.) were extracted using EasyPure Bacteria Genomic DNA Kit (Transgen, Beijing, China). DNAs and cDNAs were then quantified using a NANODROP 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). All extracted DNAs and cDNAs templates were stored at − 80 °C until use.