Hydroxymethylbilane synthase (HMBS) gene-based endogenous internal control for avian species

文献类型： 外文期刊

第一作者： Wang, Yaoyao

作者： Wang, Yaoyao;Zhang, Jilei;Li, Min;Shen, Qiuping;Yang, Yi;Wei, Lanjing;Zhang, Yuanyuan;Peng, Daxin;Ye, Jianqiang;Wang, Chengming;Patrick, Kelly;Gong, Jiansen;Xu, Bu;Poudel, Anil;Wang, Chengming

作者机构：

关键词： Endogenous internal control; Birds; PCR; HMBS

期刊名称：AMB EXPRESS （ 影响因子：3.298； 五年影响因子：3.427 ）

ISSN： 2191-0855

年卷期： 2020 年 10 卷 1 期

页码：

收录情况： SCI

摘要： With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 mu L reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.

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