A new multiplex PCR for the accurate identification and differentiation of serovar Gallinarum biovars Pullorum and Gallinarum.

serovar Gallinarum biovars Gallinarum and Pullorum cause severe chicken salmonellosis, a disease associated with high mortality and morbidity among chickens worldwide. The conventional serotyping and biochemical reactions have been used to identify serovars. However, the conventional methods are complicated, time-consuming, laborious, and expensive. Furthermore, it is challenging to distinguish Gallinarum and Pullorum biochemical assays and serotyping because of their antigenic similarity. Although various PCR methods were established, a PCR protocol to detect and discriminate Gallinarum and Pullorum simultaneously is lacking. Herein, a one-step multiplex PCR method was established for the accurate identification and discrimination of Pullorum and Gallinarum. Three specific genes were used for the multiplex PCR method, with the and genes being the key targets to identify and differentiate Gallinarum Pullorum, and being included as a reference gene for the genus. analysis showed that the gene is present in all serovars, except for Gallinarum, and could therefore be used for the identification of Gallinarum. A 68-bp sequence deficiency in was found only in Pullorum compared to other serovars, and this could therefore be used for the specific identification of Pullorum. The developed PCR assay was able to distinguish Gallinarum Pullorum among 75 various strains and 43 various non- pathogens with excellent specificity. The detection limit for the genomic DNA of Gallinarum and Pullorum was 21.4 pg./μL, and the detectable limit for bacterial cells was 100 CFU. The developed PCR method was used for the analysis of isolates in a chicken farm. This PCR system successfully discriminated Gallinarum Pullorum from other different serovars. The PCR results were confirmed by the conventional serotyping method. The newly established multiplex PCR is a simple, accurate, and cost-effective method for the timely identification and differentiation of Pullorum and Gallinarum.