Reference genes in E. ictaluri being studied.

Edwardsiella ictaluri is a Gram-negative bacterium causing enteric septicemia of catfish (ESC), leading to significant economic losses in the catfish farming industry. RT-PCR analysis is a powerful technique for quantifying gene expression, but normalization of expression data is critical to control experimental errors. Using stable reference genes, also known as housekeeping genes, is a common strategy for normalization, yet reference gene selection often lacks proper validation. In this work, our goal was to determine the most stable reference genes in E. ictaluri during catfish serum exposure and various growth phases. To this goal, we evaluated the expression of 27 classical reference genes (16S rRNA, abcZ, adk, arc, aroE, aspA, atpA, cyaA, dnaG, fumC, g6pd, gdhA, glnA, gltA, glyA, grpE, gyrB, mdh, mutS, pgi, pgm, pntA, recA, recP, rpoS, tkt, and tpi) using five analytical programs (GeNorm, BestKeeper, NormFinder, Comparative ΔCT, and Comprehensive Ranking). Results showed that aspA, atpA, dnaG, glyA, gyrB, mutS, recP, rpoS, tkt, and tpi were the most stable reference genes during serum exposure, whereas fumC, g6pd, gdhA, glnA, and mdh were the least stable. During various growth phases, aspA, g6pd, glyA, gyrB, mdh, mutS, pgm, recA, recP, and tkt were the most stable, while 16S rRNA, atpA, grpE, and tpi were the least stable. At least four analysis methods confirmed the stability of aspA, glyA, gyrB, mutS, recP, and tkt during serum exposure and different growth stages. However, no consensus was found among the programs for unstable reference genes under both conditions.

爱德华氏菌（Edwardsiella ictaluri）是一种革兰氏阴性菌（Gram-negative bacterium），可引发鲶鱼肠败血症（Enteric Septicemia of Catfish，简称ESC），给鲶鱼养殖业造成了显著的经济损失。逆转录聚合酶链式反应（RT-PCR）分析是定量基因表达的高效技术，但表达数据的标准化对于控制实验误差至关重要。采用稳定的参考基因（亦称持家基因，housekeeping genes）是基因表达标准化的常用策略，但参考基因的筛选往往缺乏严谨的验证。本研究旨在明确鲶鱼血清暴露及不同生长阶段下，爱德华氏菌体内最稳定的参考基因。为此，我们借助5种分析程序（GeNorm、BestKeeper、NormFinder、比较ΔCT法、综合排名法），对27种经典参考基因[16S rRNA、abcZ、adk、arc、aroE、aspA、atpA、cyaA、dnaG、fumC、g6pd、gdhA、glnA、gltA、glyA、grpE、gyrB、mdh、mutS、pgi、pgm、pntA、recA、recP、rpoS、tkt、tpi]的表达水平进行了评估。结果表明，在血清暴露条件下，aspA、atpA、dnaG、glyA、gyrB、mutS、recP、rpoS、tkt及tpi为最稳定的参考基因，而fumC、g6pd、gdhA、glnA及mdh的稳定性最差；在不同生长阶段中，aspA、g6pd、glyA、gyrB、mdh、mutS、pgm、recA、recP及tkt的稳定性最优，而16S rRNA、atpA、grpE及tpi则稳定性最差。至少有4种分析方法证实，在血清暴露与不同生长阶段下，aspA、glyA、gyrB、mutS、recP及tkt均具备稳定的表达特性。然而，在两种实验条件下，各分析程序对于不稳定参考基因的筛选结果并未达成共识。