Abi 3500xl genetic analyzer pop7

Sanger sequencing was performed on ABI 3500XL Genetic Analyzer POP7™ (Thermo Scientific) using the same specific primers described previously [22 (link)]. The raw sequence reads were edited with Finch TV v1.4 (Geospiza, Seattle, USA). The nucleotide sequences obtained from the selected NoV strains were used to search similar sequences in the NCBI genetic database using the BLAST tool (available at http://www.ncbi.nlm.nih.gov/) and then aligned using Noronet typing tools [26 (link)] (available at http://www.rivm.nlm/norovirus/typingtool). The reference strains from GenBank were randomly selected among the BLAST hits with >80% similarities on the query sequence of the NoV strains identified from this study. Phylogenetic trees were constructed by the neighbour-joining method [27 (link)] using MEGA 7 software, with 1,000 bootstrap replicates for each gene [28 (link)]. The evolutionary distances were computed using the P-distance method [29 ].

The PCR products were purified using the QIAquick^® PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, except that the final elution was done in 25 μl instead of the recommended 50 μl to increase the concentration of DNA. Purified PCR products were ligated into pJET1.2/blunt cloning vector using the ClonJET^TM PCR cloning Kit (ThermoFisher Scientific™, Waltham, MA USA), followed by transformation of JM109 E. coli competent cells (Zymo Research, Tustin, USA). Recombinant clones were confirmed by colony PCR performed in a 20 μl reaction volume consisting of 2X DreamTaq Green PCR Master Mix (ThermoFisher Scientific™, Waltham, MA USA), forward and reverse pJET primers each at 4 pmol. The cycling conditions were as follows; an initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min, then one cycle for the final extension step at 72°C for 1 min. The PCR products were analyzed by gel electrophoresis using 2% agarose in 1X TAE running buffer. Colony PCR products were purified using the QIAquick^® PCR Purification Kit (Qiagen, Hilden, Germany) following manufacturer’s protocol. Bidirectional sequencing was performed using pJET primers on ABI 3500XL Genetic Analyzer, POP7™ (ThermoFisher Scientific™, Waltham MA, USA) at INQABA Biotechnologies, South Africa.

The RT-PCR products of the amplified fragments were purified with Zymoclean™ Gel DNA recovery kit following manufacturer’s instructions. Using the same specific primers, Sanger sequencing was performed on ABI 3500XL Genetic Analyzer POP7™ (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequence reads were edited with Finch TV v1.4 (Geospiza Inc., Seattle, WA, USA). Nucleotide sequences of HAstVs obtained were compared with reference strains obtained in the NCBI GenBank using BLAST tool available at https://www.ncbi.nlm.nih.gov/blast / {accessed on 9 March 2020} followed by construction of phylogenetic tree using MEGA X software [47 (link)]. Reference strains from GenBank which were randomly selected among the BLAST hits had ≥80% similarities with the query sequence of the strains identified. The Neighbor-Joining method [48 (link)] was used to build the phylogenetic tree and the reliability of different phylogenetic groupings was evaluated by bootstrap analysis (1000 replicates) [49 (link)]. Evolutionary distance was computed using the p-distance method [50 ].