Automated sequencer

To detect specific mutations in the frxA, rdxA, gyrA, gyrB, and 23S rRNA genes, a PCR-based sequencing approach was carried out for a selected number of H. pylori isolates, including the susceptible and resistant strains. The oligonucleotide primers are shown in Table 1. The PCR products were sequenced on both strands by the Sanger sequencing method using an automated sequencer (Macrogen, Seoul, Korea). All complete and partial DNA sequences were edited by Chromas Lite version 2.5.1 (Technelysium Pty Ltd, Australia). Comparative sequence analysis between resistant and sensitive strains was carried out using BioEdit software version 7.2.5 [36 ]. The DNA and deduced amino acid sequences were aligned and coordinated to H. pylori 26695 (GenBank: CP003904.1) as a reference sequence.

For DNA sequencing of cagI and cagN, PCR amplification was carried out in a final volume of 25 µl using designed specific primers including 5′-CATTTGACTTACCTTGATTAC-3′ (cagI-F) and 5′-TTTGAGCACTTGTTGGTTGG-3′ (cagI-R), 5′-GAGCGACAAAACAACTATGC-3′ (cagN-F) and 5′-GATCCCTAGAACAAAGTAAGC-3′ (cagN-R) yielding DNA fragments of about 1377 and 1192 bp in length, respectively. The PCR products were purified using the Silica Bead DNA Gel Extraction Kit (Thermo Scientific, Fermentas, USA) followed by sequencing on both strands using an automated sequencer (Macrogen, Seoul, Korea). DNA sequences were edited by Chromas Lite version 2.5.1 (Technelysium Pty Ltd, Australia) and BioEdit version 7.2.5²⁹ . The cagI and cagN nucleotide and amino acid sequences were aligned to H. pylori strain P12 as a reference strain (GenBank: CP001217.1). The single nucleotide variations and codon usage of the sequences were examined using BioEdit version 7.2.5.

The bacterial isolate (HAL1) with the highest alkaline protease production was identified based on its morphological and biochemical characteristics as described in Bergey’s manual of determinative bacteriology [24 ]. The isolate was further identified using 16 s rDNA sequence analysis. Genomic DNA was extracted using the Hipura Bacterial DNA Kit (Angen Biotech, China) according to the manufacturer’s instructions. PCR amplification of the 16 s rDNA was carried out using the forward primer: 16F 27 (5′-AGA GTT TGA TCC TGG CTC AG-3′), and the reverse primer: 16R 1525 (5′-AAG GAG GTG ATC CAG CCG CA-3′). The PCR product was purified using the QIA quick gel extraction kit (Qiagen, USA) and sequenced using an automated sequencer (Macrogen, Korea). The identity of the isolate was determined by aligning the obtained sequence with the reference sequences available on the NCBI homepage using the BLAST algorithm (www.ncbi.nlm.nih.gov/blst). Multiple alignments and phylogenetic tree construction were performed using the Neighbor-Joining method using Mega-X software, version 10.1.7 [25 (link)].

Genomic DNA was extracted from the sampled tissue from each individual following the protocol described in Martín-Torrijos et al. [39 (link)]. Mitochondrial 16S and COI genes were used as markers in this study for detecting intraspecific genetic variation in crayfish. The primers pair used to amplify the mitochondrial 16S gene, 1472 [54 (link)] and Tor12sc [55 (link)], amplified a fragment that included partial sequences of the 12S rRNA, the 16S and the val-tRNA. The primer pairs used to amplify the mitochondrial COI gene was C/N 2769 [56 (link)] and LCO1490 [57 ]. Both primers pairs were used in a single round PCR following the protocols in Matallanas et al. [38 (link)], including negative and positive controls. Three microliters of aliquots of the amplification products were analyzed to check positive amplicons in 1% agarose TAE gels stained with SBYR1Safe (Thermo Fisher Scientific, Waltham, MA, USA). The amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany). Double-stranded PCR products were sequenced using an automated sequencer (Macrogen, Spain). Sequences were assembled and edited using the program Geneious v6.14 8 [58 (link)], and a BLAST search was run on each sequence to confirm they belonged to WCC species.

Genomic DNA was extracted from the sampled tissue from each individual following the protocol described in Martín-Torrijos et al. [39 (link)]. Mitochondrial 16S and COI genes were used as markers in this study for detecting intraspecific genetic variation in crayfish. The primers pair used to amplify the mitochondrial 16S gene, 1472 [54 (link)] and Tor12sc [55 (link)], amplified a fragment that included partial sequences of the 12S rRNA, the 16S and the val-tRNA. The primer pairs used to amplify the mitochondrial COI gene was C/N 2769 [56 (link)] and LCO1490 [57 ]. Both primers pairs were used in a single round PCR following the protocols in Matallanas et al. [38 (link)], including negative and positive controls. Three microliters of aliquots of the amplification products were analyzed to check positive amplicons in 1% agarose TAE gels stained with SBYR1Safe (Thermo Fisher Scientific, Waltham, MA, USA). The amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany). Double-stranded PCR products were sequenced using an automated sequencer (Macrogen, Spain). Sequences were assembled and edited using the program Geneious v6.14 8 [58 (link)], and a BLAST search was run on each sequence to confirm they belonged to WCC species.