2.3. Extraction of Genomic DNA and Detection of NHPB by PCRGenomic DNA from lobster hepatopancreas was extracted by a commercial selleck bio kit (Wizard SV Genomic, Promega, Madison, WI) following the manufacturer’s specifications. Detection of NHPB-DNA was performed by PCR with primers NHPF2 5��-CGT TGG AGG TTC GTC CTT CAG T-3�� and NHPR2 5��-GCC ATG AGG ACC TGA CAT CAT C-3�� [10]. The reaction mixture contained PuRe Taq Ready-To-Go PCR beads (GE Healthcare, USA), 0.5��L of each primer (100ng/��L), 23��L of water, and 2��L of DNA template for reaction. The cycling parameters were performed under the following conditions: 95��C/2min, 25 cycles of 95��C/30s, 60��C/30s, 72��C/30s, and final extension at 60��C/1min and 72��C/2min.PCR products were run in a 1.

2% agarose gel, and the amplification products were visualized under UV light using the KODAK Imaging System program 4.0.The PCR products were purified using a PCR purification kit QIAquick (QIAGEN, USA) following the manufacturer’s specifications; thereafter, the purified samples were prepared and sent to a specialized laboratory (CISEI) to be sequenced. Nucleotide sequences were compared with the sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”U65509″,”term_id”:”1737125″,”term_text”:”U65509″U65509 GenBank in the algorithm Blast N of the National Center for Biotechnology Information Bethesda, MD (http://www.ncbi.nlm.nih.gov/BLAST/).3. ResultsAfter 15 days from the initial inoculation, NHPB was detected in both, feces and hepatopancreas, from lobsters fed by force with bacterial inoculums.

The electrophoresis of the PCR products revealed 379bp amplicons using NHPF2/R2 primers and genomic DNA (Figures (Figures11 and and2).2). The results from sequencing the amplicons positive to NHPB (379bp) from both, feces and hepatopancreas, were compared to the NHPB reference sequence of GenBank (“type”:”entrez-nucleotide”,”attrs”:”text”:”U65509″,”term_id”:”1737125″,”term_text”:”U65509″U65509) and matched 100%. Additionally, blackish spots (2�C5mm) were observed along the hepatopancreas surface; these spots were necrotized areas (Figure 3).Figure 1Agarose gel electrophoresis analysis of PCR amplification of extracted DNA from lobster feces. PCR was performed using Primers NHP/F2 and NHP/R2 [9]. Lane M indicates low-mass DNA marker, Lane 1 feces from control lobster, Lanes 2, 3, and 5 feces from …Figure 2Agarose gel electrophoresis analysis of PCR amplification Anacetrapib of extracted DNA from lobster hepatopancreas. PCR was performed using Primers NHP/F2 and NHP/R2 [9]. Lanes 1 and 2 indicate hepatopancreas from lobsters inoculated with NHPB, Lane 3 positive control …Figure 3Hepatopancreas from lobster (H. americanus) experimentally infected with NHPB from penaeid shrimp (L. vannamei).