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

Authors
  • Wang, Yaoyao1
  • Zhang, Jilei1
  • Patrick, Kelly2
  • Li, Min1
  • Gong, Jiansen3
  • Xu, Bu3
  • Shen, Qiuping1
  • Yang, Yi1
  • Wei, Lanjing1
  • Zhang, Yuanyuan1
  • Peng, Daxin1
  • Ye, Jianqiang1
  • Poudel, Anil4
  • Wang, Chengming1, 4
  • 1 Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, China , Yangzhou (China)
  • 2 Ross University School of Veterinary Medicine, Basseterre, West Indies, St. Kitts and Nevis , Basseterre (St. Kitts & Nevis)
  • 3 Chinese Academy of Agricultural Sciences, Yangzhou, Jiangsu, China , Yangzhou (China)
  • 4 Auburn University, Auburn, Alabama, USA , Auburn (United States)
Type
Published Article
Journal
AMB Express
Publisher
Springer Berlin Heidelberg
Publication Date
Oct 07, 2020
Volume
10
Issue
1
Identifiers
DOI: 10.1186/s13568-020-01112-5
Source
Springer Nature
Keywords
License
Green

Abstract

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 µL 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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