Development And Application Of Fluorescence PCR Methods For Rapid Detection Of Salmonella

Salmonella remains a major cause of foodborne illness in humans worldwide. Traditional methods for isolating and identifying Salmonella take approximately 4-7 days. Development of rapid detection assays for Salmonella would enable official agencies and food industries to identify contaminated foodstuffs and other samples in a timely manner.More recpntly, a number of alternative methods for detection of Salmonella have been developed including immunoassays, nucleic acid hybridization and polymerase chain reaction (PCR) techniques. The PCR has become a powerful and increasingly popular tool in Salmonella identification, though it has many disadvantages such as PCR product contamination and low efficiency. However, the application of fluorescence PCR (FPCR) assay will overcome the problems above.Using the molecular beacon and the TaqMan-MGB probe, two FPCR assays were developed for the detection of Salmonella in food and other samples and used in the clinical trial and the investigation of the epidemiology of Salmonella in Shenzhen. The results are as followed.1. Development of the FPCR assays for Salmonella detection(1) Six kinds of enrichment medium were compared and the selenite-cystine (SC) was selected as the best one for FPCR assay. Four protocols were compared for the preparation of template from culture before FPCR assays and the protocol including boiling and centrifuging the enrichment media was the best.(2) Four pairs of primers were designed according to the sequence of invA. A lot of Salmonella and non-Salmonella rsdlations were screened by the four pairs of primers and the primer of Rahn, and then one pair ofthe new primers were selected for the higest specificity.(3) A TaqMan-MGB probe was designed according to the amplification fragment sequence with the best pair of primer. The amplification conditions were optimized and the TaqMan-MGB assay was established to detect Salmonella.(4) A molecular beacon was designed according to the amplification fragment sequence with the best pair of primer. The amplification conditions were optimized and the molecular beacon assay was established to detect Salmonella.2. Evaluation ofthe established FPCR assays for the detection of Salmonella(1) Specificity: 200 Salmonella were proved to be positive while 200 non-Salmonella strains were proved to be negative by both the TaqMan-MGB assay and the molecularbeacon assay.(2) Sensitivity: The detection limit of TaqMan-MGB assay (40 cycles) was 69fg/PCR for S.typhimurium DNA, 2cfu/PCR for S.typhimurium culture and 2cfu/25g for food sample. The detection limit of molecular beacon assay (40 cycles) was 364fg/PCR for S.typhimurium DNA, 2cfu/PCR for S.typhimurium culture and 2cfu/25g for food sample.(3) Anti-interference: non-Salmonella strains don't interfere with the fluorescence PCR assay. Food samples treated by recommended method will give good result.(4) Reproducibility: Parallel experiments can make consistent results. The reproducibility is good.3. Application of the FPCR assays for the detection of Salmonella(1) More 33 samples can be detected by FPCR assays than traditional method in 200 raw food samples.(2) 3 positive specimens can be detected by both FPCR assays and traditional method in 300 food-makers specimens. The coherence is 100%.(3) 28 positive samples can be detected by both FPCR assays and traditional method in 45 patient specimens. The coherence is 100%.Molecular beacon and TaqMan-MGB assay for the detection of Salmonella have been established in the study. The assays can be used in rapid detection of Salmonella in foodborne diseases and foodstuffs contamination, which can be the supplement of traditional method. They can be applied to the clinical analysis and food safety monitoring, which can increase the accuracy and sensitivity of current methods.