The assays were conducted with pure, serologically characterized strains for further studies. Supplementary Table S1 lists the Salmonella and non-Salmonella pathogens utilized to develop and validate the multiplex PCR technique. A total of 75 Salmonella isolates including 29 different serovars and 50 non-Salmonella pathogens were obtained in routine laboratory work and used in the present study. Rapid agglutination diagnostic antisera (Tianrun Bio-Pharmaceutical, Ningbo, China) were used for serotyping all identified strains of Salmonella. The biovars Gallinarum and Pullorum were distinguished from one another with the aid of fermenting dulcitol and decarboxylating ornithine.

Frozen stocks of the isolates were recovered in Luria-Bertani (LB) agar (Oxoid, Basingstoke, UK) or brain heart infusion (BHI) agar (Becton, Dickinson and Company, Sparks, MD, United States) for 18 h at 37°C for DNA purification. The colonies were inoculated into LB or BHI broth and incubated overnight at 37°C with continuous shaking at 180 rpm.

Following the protocol outlined by the manufacturer of a DNA extraction kit (Tiangen, Beijing, China), genomic DNA was isolated from the cultured bacteria. Afterward, the DNA was resuspended with 100 μL of double distilled sterile water. A NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE) was utilized to analyze the level and purity of the extracted genomic DNA. The extracted DNA was stored at−20°C in preparation for the PCR tests.

The specificity of two candidate genes, torT, and I137_14430, was examined to establish an easy-to-use, quick, and reproducible approach for identifying and distinguishing S. Gallinarum and S. Pullorum. The genes torT (GenBank acc. no. AM933173.1, region 3797861-3798901) and I137_14430 (GenBank acc. no. CP006575.1, region 3085007-3085765) were identified by screening the database for non-redundant nucleotide collection (nr/nt) utilizing the basic local alignment search tool (BLAST). All other settings were left at their defaults except for the upper limit on how many aligned sequences to be displayed, which was set at the maximum of 5,000. Sequence alignments of I137_14430 and torT from S. Gallinarum, S. Pullorum and other serovars were conducted by Clustal W. BLAST search was conducted using S. Pullorum torT, I137_14430 and stn nucleotide sequences against the torT or genome sequences of Citrobacter freundii strain FDAARGOS_549 (GenBank accession no. NZ_CP033744.1).

Three sets of primers of torT, I137_14430 and stn were designed. The positions of the primers were based on the deficient region of torT, the unique sequence of I137_14430, and the conserved sequence of stn for Salmonella genus. The first primer set, troT-F/R, was designed to amplify an 801 bp fragment to allow for the specific identification of S. Pullorum. The second primer set, I137_14430-F/R, was designed to distinguish S. Gallinarum from other strains, while the third primer set, stn-F/R, was used as a reference gene to identify Salmonella genus (Table 1). The specificity of primers was assessed by using the BLAST and primers were synthesized commercially by GenScript (Nanjing, China).

PCR tests were conducted in a 25 μL reaction solution composed of 12.5 μL of 2 × Taq Master mix (Vazyme, Nanjing, China), primer concentrations including 80 nM stn, 40 nM torT, and 40 nM I137_14430, genomic DNA from bacteria (100 ng), and highly purified water was added till the total amount reached 25 μL. PCR amplification was conducted in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) with an initial denaturation of 94°C for 3 min, 25 cycles of 94°C for 45 s, 60°C for 30 s, and 72°C for 60 s, followed by a final extension at 72°C for 10 min. Following electrophoresis, the PCR products were examined on a 1% agarose gel before staining with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA), and subsequent visualization under UV light using a GelDoc XR Gel Documentation System (Bio-Rad).

Genomic DNA was taken from 75 Salmonella strains representing 29 distinct serovars and 50 non-Salmonella pathogens presented in Supplementary Table S1, in order to test the specificity as well as the compatibility of the primer sequences in the established multiplex PCR. Specificity of the one-step multiplex PCR for the detection and differentiation of S. Pullorum and S. Gallinarum was cross-validated in another laboratory. Two strains were randomly selected for each Salmonella serotype and non-Salmonella pathogens.

To determine the limit of detection in the PCR test, its sensitivity was assessed. S. Pullorum strain S06004 and S. Gallinarum strain SG9 were grown overnight in the LB medium and the DNA was extracted with a bacterial genomic DNA extraction kit. The genomic DNA was consecutively diluted from 27.5 ng/μL to 2.75 pg/μL in sterile water and served as the templates for the multiplex PCR. Two washes with PBS were used to rinse the bacterial culture of S. Gallinarum and S. Pullorum, adjusted to match the final concentration levels of 2 × 10⁷ to 2 × 10³ CFU/mL, and DNA from bacterial genomes were extracted by boiling for 10 min in a water bath. Eventually, 5 μL of each concentration recovered via centrifugation was utilized for this PCR test to determine the lowest number of S. Pullorum and S. Gallinarum cells.

The clinical Salmonella strains were isolated from dead eggs obtained from a poultry farm in Jiangsu, China. Salmonella was isolated from samples by following previously established procedures for sample processing, enrichment, and isolation (22, 23). Briefly, all the samples underwent pre-enrichment in 50 mL of buffered peptone water (Difco, BD, Sparks, MD, United States) for 24 h at 37°C. After being streaked over xylose lysine tergitol 4 (Difco, BD) agar, the bacterial culture was subjected to incubation for 16 h at 37°C. The established multiplex PCR approach was employed to detect the DNA from the putative Salmonella colonies. Moreover, each sample was also subjected to the standard bacterial culture procedures as well as the traditional serum agglutination test.

Turkeys, chickens, and a few other types of birds are susceptible to two different strains of Salmonella, called Gallinarum and Pullorum, which correspondingly cause fowl typhoid and Pullorum disease (8). Since S. Pullorum and S. Gallinarum are both members of the same serovar yet belong to separate biovars, biochemical features are the primary basis for identifying and distinguishing between them. Even though biochemical identification is the most common method, analyzing a large number of samples rapidly may be difficult, expensive, and time-consuming. Consequently, DNA-based approaches, particularly PCR-based methods, were required for distinguishing between the two closely related biovars. Molecular screening with the use of PCR is the most time-efficient method, as it has both a high degree of specificity and sensitivity and could be applied for the prompt detection and characterization of particular types of pathogenic microbial infection (24, 25).

The selection of the target is one of the most crucial aspects involved in the development of this kind of detection assay. Based on the bioinformatics analysis, we found that a 126 bp-region of deletion in the carboxyl-terminal of torT was observed only in S. Pullorum and not in other Salmonella serovars including S. Gallinarum, which may be employed to accurately identify S. Pullorum (Figure 1A; Supplementary Figure S1). The I137_14430 gene, which is found in all Salmonella serovars besides S. Gallinarum, could be utilized to identify this biovar (Figure 1B; Supplementary Figure S2). The similarity and existence of the three targets were also examined in C. freundii. The results show that the length of S. Pullorum torT gene is 923 bp. However, the length of C. freundii torT gene is 1,032 bp. Besides, no significant similarity was found in the torT sequences between the two species (Supplementary Figure S3A). I137_14430 and stn genes of S. Pullorum were aligned with the genome of C. freundii, and no significant similarity was found in C. freundii genome for both I137_14430 sequence (Supplementary Figure S3B) and stn sequence (Supplementary Figure S3C). Furthermore, the gene stn is extensively employed as the reference control of the Salmonella genus (26–28). Thus, based on the sequence characteristics, three sets of primers of torT, I137_14430, and stn were designed. The three pairs of primers amplified three specific products for torT (801 bp), I137_14430 (508 bp), and stn (252 bp) (Table 1).

Novel gene-based tests for identifying Salmonella have also been published. For instance, by examining the whole genome sequencing data of S. Pullorum serotypes, Xu et al. (29) effectively designed the PCR test for the recognition of S. Pullorum predicated on the novel gene ipaJ of S. Pullorum. However, it could not differentiate the two biovars of S. Gallinarum and S. Pullorum. Notably, our research successfully identified and differentiated S. Pullorum and S. Gallinarum for the first time by targeting the new genes of torT and I137_14430.

Salmonella needs to be identified and serotyped to offer additional details that can be used for the determination of the sources of infection during outbreak investigations and for strain identification (30). Nevertheless, the findings of the majority of genotyping techniques, such as plasmid profile analysis, pulsed-field gel electrophoresis (PFGE), ribotyping, and amplified fragment length polymorphism (AFLP), do not show a correlation between the genotype and the serotype of the Salmonella serogroup (31).

To evaluate the specificity of the three primers sets, the multiplex PCR was first optimized and then tested with DNA templates prepared from the 50 non-Salmonella and 75 Salmonella pathogens as listed in Supplementary Table S1. The results showed that only two specific products of 508-bp I137_14430 and 252-bp stn were amplified for S. Pullorum. Only two products of 801-bp torT and 252-bp stn were generated for S. Gallinarum. All three products of torT, I137_14430, and stn were amplified for other Salmonella serovars. Nonetheless, there was not a single band generated for any of the pathogens that were not Salmonella (Figure 2). The three specific targets could not be amplified in four strains of C. freundii preserved in our laboratory using the designed primers in this study (Figure 3), which was consistent with those of bioinformatics analysis. Cross validation of tests between laboratories was carried out to verify the accuracy of the multiplex PCR assay. Two strains were randomly selected for each Salmonella serotype and non-Salmonella pathogens (including Citrobacter spp.). The results were consistent with those in our laboratory, and this multiplex PCR method could accurately identify and distinguish S. Gallinarum and S. Pullorum (Supplementary Figure S4), showing that this method has good reproducibility. Additionally, there were no false positives or negatives created in the established PCR method using the three pairs of specific primers. Meanwhile, the multiplex PCR did not indicate any cross-reaction with 100% specificity, in line with the BLAST results. This PCR test had excellent specificity and could effectively identify and differentiate between S. Pullorum and S. Gallinarum.

Traditionally, S. Pullorum was distinguished from S. Gallinarum utilizing approaches like PCR restriction fragment length polymorphism (RFLP) and single-strand conformational polymorphism (SSCP) which employ changeable sections of a gene or a single-nucleotide polymorphism (SNP) (32, 33). This research, however, used the torT and I137_14430 genes to definitively tell S. Pullorum and S. Gallinarum apart. Moreover, as the primers of the torT gene are specific for S. Pullorum, and the primers of I137_14430 are specific for S. Gallinarum, it would be possible to distinguish the two biovars utilizing the two specific targets independently. Significant time and labor savings are possible since the established multiplex PCR can amplify specific DNA sequences and distinguish between the two Salmonella biovars simultaneously.

Two distinct types of templates were used to determine the sensitivity of the multiplex PCR. The detection limit of the PCR technique was evaluated by serially diluting S. Gallinarum and S. Pullorum genomic DNA from 27.5 ng/μL to 2.75 pg/μL. It was determined via sensitivity testing that a minimum of 27.5 pg/μL of genomic DNA was needed for the identification of S. Pullorum and S. Gallinarum following the multiplex PCR detection (Figure 4A). The sensitivity of the developed multiplex PCR is higher than that of the HRM-PCR test, which has a sensitivity of 126.2 pg/μL (34).

Moreover, a ten-fold serial dilution of S. Pullorum and S. Gallinarum cells that ranged from 10⁵ CFU to 10¹ CFU was used to determine the detectable limit of bacterial cells. The results from three primer sets confirmed that 100 CFU was the lowest concentration at which S. Gallinarum and S. Pullorum could be detected (Figure 4B). These PCR results showed a lower threshold for bacterial cell recognition than those from the sefA gene-based PCR (400 CFU) (35), comparable to the multiplex PCR centered on flhB, lygD, and tcpS genes (100 CFU) (36), and the PCR based on the ipaJ gene (100 CFU) (29). These findings confirmed that the PCR technique had a superior limit of detection, allowing for the identification of S. Pullorum and S. Gallinarum cells at very low quantities. Besides, this PCR assay was also very rapid, with a single-round PCR procedure taking < 2 h.

Both PD (which is caused by S. Pullorum) and FT (which is caused by S. Gallinarum) have a tendency to cause severe economic losses of livestock (37). S. Pullorum and S. Gallinarum are two of the most significant bacterial infections that affect chicken in China (38). The accurate identification and subsequent removal of diseased birds are essential for the prevention and success in eliminating S. Gallinarum and S. Pullorum in poultry.

To assess the diagnostic performance in terms of the accuracy of our multiplex PCR approach, further clinical samples obtained from naturally contaminated chicken egg specimens were used. The findings of the PCR proved that ten of the twenty-four samples only included the unique 508-bp target of I137_14430 and the 252-bp target of stn, which implied that the isolates of Ch4, Ch5, Ch9, Ch10, Ch11, Ch12, Ch16, Ch17, Ch18 and Ch24 were S. Pullorum. Both the unique 801-bp torT and 252-bp stn were generated by two different isolates, confirming that the two isolates of Ch22 and Ch23 were S. Gallinarum. The outcomes of the PCR experiment were consistent with the findings obtained using traditional biochemical reactions and serotyping techniques (Table 2).

Classical microbiological techniques are proven to be less efficient and less sensitive than PCR-based testing in identifying Salmonella serovars (5). Molecular techniques, like PCR accompanied by RFLP predicated on the speC or fliC genes, have been utilized to distinguish between S. Gallinarum and S. Pullorum to identify these infections (10, 32). Other methods, such as a duplex PCR and a multiplex real-time PCR, have been designed to tell apart between S. Pullorum and S. Gallinarum (2, 39). However, these methods were expensive and required special equipment. The multiplex PCR established in this study is a perfect fit for this need, and it offers technological assistance for eliminating the serovar Gallinarum in commercial poultry farms.

These findings validated torT, I137_14430, and stn genes as promising candidates for S. Gallinarum and S. Pullorum identification and differentiation. Salmonella serotyping using the traditional serological method is time-consuming, labor-intensive, and expensive. The technology developed in this study, which is based on multiplex PCR, makes it possible to distinguish S. Gallinarum from S. Pullorum rapidly and accurately. As poultry may also get infected with S. Enteritidis, S. Typhimurium, and other serovars, the multiplex PCR may be able to differentiate between S. Gallinarum biovars and other Salmonella serovars. This test might enable early diagnosis of infections, allowing for more efficient disease management in poultry as well as earlier detection of infections.