DNA of selected LAB was extracted using a DNA extraction kit (QIAamp Blood and Tissue Kit, Qiagen, Hilden, Germany). The amplification of the 16SrRNA genes was conducted using 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R 5′-GGCTACCTTGTTACGACTT-3′primers. PCR amplification was performed with i-StarTaq DNA polymerase kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea) using 1 μL of a template (10 ng/μL) in 20 μL of the reaction solution. Amplification was performed using a BIORAD MyCycler™ thermal cycler with the following program: 1 cycle at 94 °C for 4 min, 30 cycles of 94 °C for 1 min, 55 °C for 30 s, 72 °C for 2 min and a final extension at 72°C for 5 min. The PCR products were mixed with loading dye and loaded on to a 1.0% SeaKem® GTG® agarose (FMC BioProducts, Rockland, ME, USA) containing ethidium bromide, and electrophoresis was carried out at 90 V for 1 h. The PCR products were visualized under UV illumination, excised from the gel, and the PCR product was extracted using MEGA quick-spin PCR & Agarose Gel Extraction kit (iNtRON Biotechnology). The PCR product was sequenced using forward and reverse primers (1st Base Co., Malaysia). The amplified products were analyzed by Sanger sequencing using the ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). The contig was done for the forward and reverse sequences of each isolate by the contig assembly program of Bioedit v. 7.2.0 software [15] and then sequences were analyzed by the Bellerophon [16] and Mallard program [17] to remove chimeric rDNA clones. Approximately 1400 bp segments of the 16S rRNA gene of the isolates were subjected to analysis by BLAST using National Center for Biotechnology Information (NCBI) library with the following address: http://blast.ncbi.nlm.nih.gov/Blast.cgi, 21 February 2020. The isolates were identified according to the BLAST results and the 16S rRNA gene sequences submitted to NCBI GenBank. The obtained accession number were as follow: L. plantarum (KJ160209), L. salivarius (KJ160204), L. reuteri (KJ160196), L. brevis (KJ160214), S. bovis (KJ160185). All identified lactic acid bacteria were from the cecum of broiler chickens except for S. bovis, which was isolated from rumen samples.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.