A one-step closed-tube enzyme-activated blocked probe assay base on single nucleotide polymorphism for rapid detection of Salmonella Pullorum

Abstract Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) is an infectious bacterial pathogen in the poultry industry that causes systemic Pullorum disease. This disease causes great losses in terms of the clinical production and quality of chicken products in breeding farms. However, an acknowledged usable rapid detection method for its specific identification has not been reported, and it is generally difficult to distinguish from fowl typhoid caused by Salmonella enterica serovar Gallinarum biovars Gallinarum. The development of a specific and rapid detection method for this pathogen is therefore needed. In the present study, we targeted the single nucleotide mutation position 237 of the S. Pullorum rfbS gene to develop an enzyme-activated blocked probe (EA-probe) for its clinical rapid detection. The method displayed robust specificity and reproducibility, and it achieved minimal detection limits of 21 copies/μL of copy number and 4.53 pg/μL of genomic DNA. Compared with traditional identification and PCR methods, this method performed better for the detection of 100 clinical actual samples, and without false negative results. The entire process can be accomplished in a one-step closed-tube operation, overcomes the difficulties currently associated with S. Pullorum detection, and provides a specific and rapid method with broad application potential for single nucleotide polymorphism detection.