Detection Of Enterbacter Sakazakii (Cronobacter Spp.) From Powdered Infant Formula Through Real-time PCR

Enterbacter sakazakii is a kind of parasitic in humans and animals intestinal gram-negative bacillus,general flagella,can exercise,facultative anaerobic.This bacterium can lead to serious conditions such as meningitis,necrotizing enterocolitis and bacteremia,.They can affect the crowd of all ages who have low immunity,especially the newborn babies,preterm,babies born with poor physical condition.After infection,infant mortality rate is as high as 500h above.This is a kind of important food-borne pathogens.In recent years,a growing number of domestic and foreign studies have suggested that the infant formula milk powder is the main source for the spread of infant infection and transmission medium.At present,the GB method to detect Enterbacter sakazakii is largely based on the traditional microbial cultures,and then identify in combination with colony morphology,biochemical characteristics,etc.This operation is complicated,time-consuming,and not easy to distinguish a result,there are some false positives.So in recent years,with the method of molecular biology detection of foodborne pathogens are more and more widely used.This paper is based on the entry and exit inspection and quarantine of the People's Republic of China industry standard(SN/T 1632.3 2013)of Enterbacter sakazakii(Cronobacter spp)real-time fluorescent quantitative PCR testing method.Firstly,PCR reaction components are mixed together called Mix Buffer,which greatly simplifies the operation steps.At the same time,each component concentration and reaction system has been optimized,so that rapid detection of Enterbacter sakazakii with Real-time PCR is realized.Secondly,by comparing the two methods of DNA extraction by phenol chloroform and heating pyrolysis,the method of heating pyrolysis is optimized,and is verified using the milk powder samples and artificial contaminated milk powder samples.The detection limit of artificial contaminated milk powder with Enterbacter sakazakii is 4.7 x 103 cfu/ml.Real-time PCR experiments in this paper are completed on the real-time fluorescent quantitative PCR instrument platform which is developed by Bioer company and in the standardized PCR lab which is Bioer's patent products.Enterbacter sakazakii rapid detection system which is composed of reagents,equipment,software,PCR pollution prevention and control is established preliminarily.The system provide total solution for the customer from hardware to software,from the experiments to environmental pollution control.The stability of rapid detection is realized.