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Salmonella sp. specific gene fragment 3335471 was used to establish a PCR-based assay for rapid detection of Salmonella sp. To establish the PCR assay, internal amplification control (IAC) was constructed using the composite primer method; the optimal concentration was determined to be 365 copies/μL. Storage of freeze-dried PCR solutions at –20°C for 3 months had no effect on detection results. Tests of artificially contaminated milk showed that Salmonella choleraesuis could be detected in 8 h when inoculated at 7 colony forming units (CFU)/10 mL. Next, a quantitative real-time PCR (RT-PCR) for detection of Salmonella sp. was developed. The assays showed high specificity for 13 Salmonella strains and 16 non-Salmonella strains. The optimal concentration of the IAC was 425 copies/μL for RT-PCR. Using S. choleraesuis as the target strain, the detection sensitivity of genome was 1.23 fg/μL and the limit of detection was 1–4 CFU/10 mL in artificially contaminated milk with 6 h of non-selective enrichment. This study established highly efficient, sensitive PCR and RT-PCR systems, providing effective approaches for the rapid detection of Salmonella sp. in the food industry.

In this study, we developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Salmonella detection targeting the gene62181533. The addition of manganous ions and calcein to the reaction system allowed the visualization of an apparent color change during the amplification reaction. The detection limit of LAMP using a six-primer combination was 1.057 copies/μl (standard plasmid), 4.1 CFU/ml (cell), and 7.68 fg/μl (genome). The LAMP assay was 100% specific based on 32 Salmonella strains and 25 non-Salmonella bacteria. In inoculated milk, the LAMP assay detected Salmonella at an initial inoculation concentration of 0.81 CFU/ml following 12 h of pre-enrichment in buffered peptone water. One hundred food samples (beef, chicken, and pork) were tested by the LAMP assay. The positive-sample ratios were 11/100 by LAMP, 11/100 by conventional PCR, and 11/100 by a culture-based method. The results revealed that the gene62181533-based LAMP assay is a rapid, specific, and sensitive method for the detection of Salmonella in foods.

The purpose of this study is to find new molecular targets for the detection of Salmonella. With the online BLAST Program, we compared homology of genomic sequences and specificity in GenBank among Salmonella serovars and non-Salmonella strains and found 98 Salmonella specific target sequences. We selected 33 target sequences of Gene ID from 3335000 to 3337003 for the specificity evaluation, and finally 8 specific fragments screened out, they are 3334138, 3335583, 3335471, 3335211, 3335068, 3336466, 3336736 and 3336998. Primer SC8 of gene 3335583 and SC9 of gene 3335471 were the best in specificity and sensitivity among these primers. The detection sensitivity of Primer SC9 was 1.23 fg/μl for DNA templates and 720 cfu/ml for whole cells, while primer SC8's was 12.3 fg/μl and 720 cfu/ml, respectively. Salmonella could be detected successfully by the PCR method developed in this study after 8 h enrichment when the milk samples were artificially contaminated by this organism at 7 cfu per 10 ml milk.

Salmonella enterica serotype Paratyphi B is a globally distributed human-specific pathogen causing paratyphoid fever. The aim of this study was to develop a rapid and reliable polymerase chain reaction (PCR) assay for its detection in food. The SPAB_01124 gene was found to be unique to S. Paratyphi B using comparative genomics. Primers for fragments of the SPAB_01124 gene and the Salmonella-specific invA gene were used in combination to establish a multiplex PCR assay that showed 100% specificity across 45 Salmonella strains (representing 34 serotypes) and 18 non-Salmonella strains. The detection limit was 2.2 CFU mL(-1) of S. Paratyphi B after 12-h enrichment in pure culture. It was shown that co-culture with S. Typhimurium or Escherichia coli up to concentrations of 3.6 × 10(5) CFU and 3.3 × 10(4) CFU, respectively, did not interfere with PCR detection of S. Paratyphi B. In artificially contaminated milk, the assay could detect as few as 62 CFU mL(-1) after 8 h of enrichment. In conclusion, comparative genomics was found to be an efficient approach to the mining of pathogen-specific target genes, and the PCR assay that was developed from this provided a rapid, specific, and sensitive method for detection of S. Paratyphi B.