Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae

文献类型： 外文期刊

第一作者： Li, Shiyang

作者： Li, Shiyang;Deng, Zhibang;Yu, Yanfei;Yuan, Xiaomin;Li, Shiyang;Zhou, Yanqing;Yuan, Ting;Feng, Zhixin;Zhang, Zhenzhen;Wu, Yuzi;Xie, Qingyun;Wang, Jia;Yu, Yanfei;Zhou, Yanqing;Feng, Zhixin;Yu, Yanfei;Zhou, Yanqing;Li, Quan;Yu, Yanfei

作者机构：

关键词： Mycoplasma hyopneumoniae; RT-qPCR; real-time quantitative polymerase chain reaction; internal reference genes; gatB; virulence

期刊名称：FRONTIERS IN VETERINARY SCIENCE （ 影响因子：3.471； 五年影响因子：3.821 ）

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年卷期： 2022 年 9 卷

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收录情况： SCI

摘要： Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.

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