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

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.

Identification of reference genes for expression studies in the liver, and spleen of laying hens housed in cage and cage-free systems

“…Previously, in magnum tissue of hens housed in CF systems, the most stable gene was HMBS, similar to our results in the spleen of hens of the CF (geNorm and NormFinder) and comprehensive data (CC-CF; BestKeeper) group ( Rodríguez-Hernández et al , 2021 ). On the other hand, the HMBS gene was reported as the most stable gene in the spleen of quail ( Macario et al , 2022 ) and codifying for HMBS protein, a vital enzyme in the heme biosynthetic pathway ( Wang et al , 2020 ).…”

Identification of stable reference genes for quantitative gene expression analysis in the duodenum of meat-type ducks

“…The above research results are consistent with the results of the experiments in this study, suggesting that HMBS may be the most suitable gene as an internal reference gene in birds. Another study mentioned that HMBS is highly specific to avian species ( 26 ). Based on this, HMBS can help reduce the possibility of false negative tests caused by insufficient sampling or storage degradation.…”

Identification of reference genes for studies of quantitative gene expression in male and female quail tissues.

“…This enzyme is involved, also, in production of molecule called heme, considered vital for all organs of the body in both males and females. The use of HMBS gene as an endogenous control for RT-qPCR analysis in various tissues of avian species has been reported and has shown positive results, due to its high expression and stability [16].…”