Bigdye terminator v3.1 cycle

The molecular identification was carried out as described by Fguiri et al. (2015) for further confirmation of isolated bacteria from the RTE chilli shrimp paste. The genomic DNA of isolated SSO of chilli shrimp paste was extracted using the DNA extraction and purification kit BigDye® Terminator v3.1 Cycle (Applied Biosystems) according to the manufacturer instructions. The bacterial 16S rDNA, full-length 1.5 kb, was amplified using universal primers 27F (5' AGAGTTTGATCMTGGCTCAG 3') and 1492R (5' TACGGYTACCTTGT-TACGACTT 3'). The total reaction volume of 25 µL contained gDNA purified using an in-house extraction method; 0.3 pmol of each primer, deoxynucleotides triphosphates (dNTPs, 400 µM each), 0.5 U DNA polymerase, supplied PCR buffer, and water. The PCR was performed as follows: one cycle (94°C for 2 min) for initial denaturation, and 25 cycles (98°C for 10 s; 53°C for 30 s; 68°C for 1 min) for annealing and extension of the amplified DNA. The PCR products were purified by standard methods, and directly sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The obtained nucleotide sequences were analysed using the blast tool of the NCBI site to obtain the identity percentages with the sequences present in the database.