Abstract
Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.
Highlights
Infectious coryza is an acute upper respiratory tract disease of chickens
The causative agent of infectious coryza is Avibacterium paragallinarum (A. paragallinarum) [2], and A. paragallinarum is classified into three serovars: A, B, and C according to the Page schemes [3]
It was notable that the gene encoding superoxide dismutase had the lowest expression levels in serovar A and C, reaching a cycle threshold after 35 amplification cycles and there was a significant difference in Cq value among serovars A, B, and C, while the mean Cq value of the sodA gene in serovar B was 24.27
Summary
Infectious coryza is an acute upper respiratory tract disease of chickens. This disease is of worldwide economic significance and affects both broiler and layer flocks, manifested primarily as a drop in egg production (10±40%) in layer flocks and retardation of growth due to decreased feed and water consumption in breeder and broiler flocks. The most common clinical signs are nasal discharge, conjunctivitis, facial oedema, lacrimation, anorexia, and diarrhea [1]. The causative agent of infectious coryza is Avibacterium paragallinarum (A. paragallinarum) [2], and A. paragallinarum is classified into three serovars: A, B, and C according to the Page schemes [3]. It is widely accepted that the three Page serovars represent distinct.
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