Identification Of New Targetsfor Detection Of Pseudomonas Fluorescens And Development Of A RCR Detective System

Pseudomonas Fluorescens is a cryophile which is advantage bacterialeading refrigerated pork and raw milk to decomposition. It is a classicconditioned pathogen that results in immunocompromised patients'Septicemia. As our living standard has improved and dietary habit has beenchanging, Pseudomonas Fluorescens becoming more and more dangerous forhuman health. The correct identification and rapid detection of this bacteriumis of great importance.PCR technology had been applied widely in the detection of foodbornepathogen for its high sensitivity, specificity and rapidness. For PseudomonasFluorescens there are limited species-specific gene targets which are lack ofsystemic evaluation.In this study 4 candidates for Pseudomonas Fluorescens-specific targetswere successfully identified by comparing the sequences of PseudomonasFluorescens and other prokaryotic genomes using bioinformatic analysis. While the 16S-23S rDNA internal transcribed spacer sequence and gyrB genewere analysed too. Primers were designed for the 6 gene targets and theirspecificity was verified by testing against 11 Pseudomonas Fluorescens and10 non-Pseudomonas Fluorescens strains. Finally according to gyrB8 genewe developed a PCR system and a real-time PCR system for detecting thePseudomonas Fluorescens.1. The PCR detection system for foodborne Pseudomonas Fluorescens wasevaluated:1) Specificity of this PCR system was tested with 11 PseudomonasFluorescens strains and 28 non- Pseudomonas Fluorescens strains. Therewas a 271-bp amplicon resulted from all Pseudomonas Fluorescensstrains, while no amplicon resulted from non- Pseudomonas Fluorescensstrains;2) Sensitivity of this PCR system were 14.3 fg genomic DNA/μL (2~3copies/μL) and 3.0×102 CFU/mL; Detection of an initial inoculum of 0.3CFU/25 mL soy milk was possible after 15-hour selective enrichment.3) Anti-interference ability of this PCR system was tested in the presence of108 CFU/ mL cells of non- Pseudomonas Fluorescens bacteria (15-hourenrichment) as background;4) 11/56 natural samples were confirmed positive by this PCR system but 1/56 was proved positive by culture methods.2. The real-time PCR detection system with a TaqMan probe forPseudomonas Fluorescens was evaluated.1) Specificity of this PCR system was tested with 11 PseudomonasFluorescens strains and 28 non- Pseudomonas Fluorescens strains.Pseudomonas Fluorescens strains generated a target fluorescent signal(Ct value＜35), while non- Pseudomonas Fluorescens strains generatednone (or Ct value＞35);2) Sensitivity of this PCR system were 14.3 fg genomic DNA/μL (2~3copies/μL) and 3.0×102 CFU/mL; Detection of an initial inoculum of 0.3CFU/25 mL soy milk was possible after 15-hour selective enrichment.3) Anti-interference ability of this PCR system was tested in the presence of108 CFU/ mL cells of non- Pseudomonas Fluorescens bacteria (15-hourenrichment) as background;4) 14/56 natural samples turned out positive by real-time PCR analysiswhich was more than end-point PCR method.In conclusion, the PCR detection system established in this study wasrapid, specific and sensitive, which is worth to be extended.