Endogenous controls of amplification in the indication of infectious diseases of cattle and small ruminants

For reliable interpretation of PCR results, it is necessary to use a series of control reactions, namely: a positive, negative and an internal control. Internal control can be of two types: exogenous, which involves adding nucleic acids to the sample being analyzed (during DNA or RNA extraction) for hybridization of primers for internal amplification control, and endogenous, where the nucleic acids for control are already present in the sample itself. This study focuses on analyzing non-species-specific genes from cattle, sheep, and goats and identifying conserved loci that can be used for internal amplification control in the detection of nucleic acids from infectious agents in cattle and small ruminants. A number of genes from cattle and small ruminants were selected for oligonucleotide design, namely: beta-galactosidase, hyaluronidase, diacylglycerol O-acyltransferase, kappa-casein, lactoferrin, neuraminidase, oxytocin, and phosphorylase kinase. Taking into account the nucleotide polymorphism of non-species-specific genes in cattle, sheep, and goats, it was possible to identify the most conserved loci of the analyzed genes for the target animals, suitable for the design of corresponding primers and probes. By comparing the nucleotide sequence differences of the entire gene and its exons for each gene, it was possible to identify the most conserved loci of the exons of two of the analyzed genes, suitable for the design of corresponding primers and probes. Aligning the nucleotide sequences for each gene revealed excessive nucleotide polymorphism across different animals when aligning the nucleotide sequence of the entire gene and its exons. However, when aligning only the exons of the analyzed genes, numerous conserved loci were observed, suitable for designing of corresponding oligonucleotides. Oligonucleotides complementary to the exons (two exons separated by an intron) of the beta-galactosidase and neuraminidase genes were proposed as internal controls for RT-PCR.

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