Rapid on-site detection for Pediococcus species based on genomic data analysis

다국어 초록 (Multilingual Abstract)

The genus Pediococcus is important lactic acid bacteria commonly found in fermented foods and beverages, contributing to flavor development and preservation. Although they have significant commercial value, their classification relies on outdated identification methods. In this study, we employed computational pangenome analysis to develop an on-site PCR method distinguishing the 10 Pediococcus species present in food products. Analysis of pan and core-genome phylogenies showed clustering of strains into 10 distinct groups according to the species. Therefore, pangenome analysis for selecting species-specific genes was applied and primer pairs that can identify ten species were designed. As a result, the high specificity (100%) using 10 Pediococcus species strains and 50 other reference strains was demonstrated. Also, the sensitivity of the developed on-site PCR approach reached 101 colony forming units (CFUs)/ml. All standard curves exhibited excellent linear relationships, with R2 values of ≥0.990. Moreover, when compared to traditional microbial identification methods, such as 16S rRNA gene sequencing and MALDI-TOF MS, the on-site PCR method exhibited superior accuracy. The applicability of the on-site PCR method was verified by monitoring 32 fermented foods or dairy products. This method has the potential to improve quality control, optimize fermentation processes, and ensure safety and consistency in food production.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 6
• 2.1. Bacterial strains and cultivation 6
• 2.2. Whole-genome sequences and ANI analysis 9
• 2.3. Pangenome analysis and extraction of species-specific genetic markers 13
• 2.4. On-site PCR conditions 14
• 2.5. Specificity and sensitivity testing of the on-site PCR 16
• 2.6. Validation of the on-site PCR method 17
• 2.7. Field application potential of the on-site PCR 19
• 3. RESULTS AND DISCUSSION 20
• 3.1. Comparative ANI analysis 20
• 3.2. Pangenome analysis and genetic marker characterization in Pediococcus species 22
• 3.3. Specificity of the on-site PCR 27
• 3.4. Sensitivity of the on-site PCR 30
• 3.5. Comparison of identification methods for Pediococcus species 33
• 3.6. Application of on-site PCR for monitoring Pediococcus species 35
• 4. CONCLUSION 37
• 5. REFERENCES 38
• 국문 초록 45