Mx3005p qpcr systems instrument

To enumerate the Acinetobacter KU011TH in the experimental systems, genomic DNA of strain KU011TH in target organs and rearing water was obtained as described above. Quantification of bacterial numbers was performed using a qPCR assay. Specific primers for the DNA gyrase subunit B (gyrB) gene of Acinetobacter KU011TH were obtained from its whole-genome sequence (accession number MG950236) and are listed in Table 1.
Samples were subjected to a qPCR assay using Brilliant III Ultra-Fast SYBR Green (Agilent, CA, USA) in an Mx3005P QPCR Systems instrument (Agilent, CA, USA). The qPCRs were previously described. The specificity and efficiency of the primers were evaluated by conventional PCR and real-time PCR, respectively. Using the above optimized real-time PCR amplification conditions, a standard curve for quantification of Acinetobacter KU011TH was prepared using 10-fold DNA serial dilutions from 1 × 10² to 1 × 10¹⁰ copies/μL. The threshold cycle (C_T) was measured for each qPCR. The obtained C_T values of the samples were then plotted against the number of microorganisms. The resulting values (DNA copy numbers) were calculated from the standard equation for a target strain. All tests and qPCRs were run in triplicate.

The immune-related genes IgM, IgT, IgD, CD4, MHCIIα, and TCRα were selected as targets in the expression analysis. IgM and IgT primers were specifically designed based on their secreted Ig forms. All the primers used in the study were validated in Asian seabass by RT-PCR amplification and nucleotide sequencing and are listed in Table 1.
The qRT-PCR assays were performed using Brilliant III Ultra-Fast SYBR^® Green (Agilent, Santa Clara, CA, USA) in an Mx3005P QPCR Systems instrument (Agilent, Santa Clara, CA, USA). The qPCRs were optimized in 15 μL reactions, including 10 μL of 2×SYBR Green QPCR Master Mix, 1 μL of 0.5 mM forward and reverse primers, 1 μL of cDNA template, and distilled water to adjust the reaction to a final volume of 15 μL. The qPCR cycling conditions consisted of an initial condition of one cycle of 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 90 s, and a final extension at 72 °C for 10 min. Triplicate qRT-PCRs were conducted for each sample. The housekeeping gene β-actin was used to standardize the results by eliminating variation in mRNA and cDNA quantity and quality. The relative expression of immune-related genes in the whole body of Asian seabass at different time points was calculated using 2^−ΔΔCT analysis following the protocol of Livak and Schmittgen (2001) [21 (link)].