tailieunhanh - Immobilization with metal hydroxides as a mean to concentrate food patthogens for increasing sensitivity of pcr based detection method

Cố định vi khuẩn bằng hydroxide kim loại làm tăng độ nhạy của phương pháp PCR khi phát hiện vi khuẩn trong thực phẩm. Vi khuẩn ở nồng độ thấp không qua nuôi dưỡng có thể phát hiện được bằng phương pháp PCR nhờ cô đặc vi khuẩn từ thực phẩm bằng hydroxide kim loại. Enzyme DNase I được sử dụng để loại bỏ DNA từ vi khuẩn sống, do đó phương pháp PCR có thể dùng để phát hiện vi khuẩn sống. SYBR green I được sử dụng trong real-time PCR để định lượng DNA được nhân lên, từ đó xác định số lượng vi khuẩn ban đầu. | Tạp chỉ Công nghệ Sinh học 5 4 437-445 2007 IMMOBILIZATION WITH METAL HYDROXIDES AS A MEAN TO CONCENTRATE FOOD PATHOGENS FOR INCREASING SENSITIVITY OF PCR BASED DETECTION METHOD Do Thi Thu Huong1 Sudip K Rakshit2 1 Agricultural Genetics Institute AGl Vietnam Asian Institute of Technology Thailand SUMMARY Polymerase chain reaction PCR based bacterial detection without enrichment step could be applicable by concentrating bacteria from food matrix by metal hydroxide. The false positive from exogenous DNA of dead cells could be eliminated by DNase I treatment 15 u 1 hour 37 C one step before DNA extraction. DNase 1 facilitated for DNA removal DNA from dead cells and had no detectable damaging effect signal on the DNA presented in live intact cells. Double-Stranded DNA dye SYBR green I was used in the real time PCR to quantify the amplicon production. In immobilization process bacterial recoveries ranged from to 99 3 for both metal hydroxides compared to with no immobilization. When seeded in 10 ml of whole milk sample volume was reduced 50 -fold after immobilization by zirconium hydroxide. The detection limits for . coli O157 H7 384 bp L. monocytogenes 482 bp E. coli wild type 580 bp was 5x1 o cells and of 5. typhimurium 685 bp was 5 x 1 o2 cells in 10 ml of whole milk by real-time PCR analysis. Keywords Bacterial recoveries DNase I immobili INTRODUCTION Food-borne pathogens such as Salmonella Escherichia coll and Listeria monocytogenes are among the most serious threats to human health. A large number of rapid methods have been developed for food-borne hazard utilizing the optical electrochemical biochemical and physical properties of microorganisms Hobson et al. 1995 . In general individual method suffers from various disadvantages that include a lengthy analysis time high cost of instrumentation and lack of sensitivity. Moreover pathogen contamination is low concentration so detection includes lengthy culture enrichment steps to increase target .