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Development of PMA-PCR Method for Detection of Viable Enterobacter sakazakii in Pasteurized Milk

LIU Yan-yan1，LIU Zeng-shan1，LU Shi-ying1，REN Hong-lin1，GAI Dong-xue1，CUI Cheng1，DING Yan-xia1，MENG Xian-mei1，2，YU Shi-yu3   

  1. （1. Key Laboratory of Zoonosis Research，Ministry of Education， Institute of Zoonosis， College of Veterinary Medicine,Jilin University, Changchun 130062，China；2.Key Laboratory of Grain and Oil Processing of Jilin Province，Biological Engineering Department，Jilin Business and Technology College，Changchun 130062，China；3.Fujian Entry-exit Insection and Quarnatine Bureau，Fuzhou 350001，China）
  • Revised:2013-12-02 Online:2014-02-20 Published:2014-03-27

Abstract: In order to detect viable Enterobacter sakazakii in pasteurized milk，a new method was established by combination of propidium monoazide （PMA） and polymerase chain reaction （PCR）.PMA concentration and exposure time were optimized to find optimal conditions for distinguishing between dead and viable E.sakazakii. The results showed that to kill the viable E.sakazakii must be exposed to 100 ℃ for 20 min in water bath. The optimum light exposure time to intercalate the DNA of dead E.sakazakii and to photolyze the free PMA in solution was 15 min. The minimum concentration of PMA to completely inhibit the PCR amplication of dead bacterium was 5 μg/mL. The maximum concentration of PMA did not inhibit the PCR amplification of viable bacterium was 15 μg/mL. After PMA treatment，viable E.sakazakii in a mixture containing different proportions of dead and viable bacterium could be selectively detected by PCR，and the determination limit was 40 CFU/mL. In the pasteurized milk，the determination limit was 100 CFU/mL. This study laid a foundation for use of PMA-PCR to detect the E.sakazakii in the food.

Key words: PMA; PCR; Enterobacter sakazakii; pasteurized milk