Detection Of Harmful Bacteria In Embryonated Egg In Southern Hebei Province

Embryonated egg and its product are popular foods,but they have a threat to people's health due to the contamination of chick embryo during incubation period.Therefore,in order to improve the health survival rate of chick embryo and the economic benefits of hatchery and ensure the safety and public health of embryonated egg products,it is essential to understand the kinds of contaminated pathogenic bacteria in embryonated egg and establish a rapid method to detect these bacteria.1.In order to investigate kinds of the pathogenic bacteria which contaminated dead embryonated egg,91 dead embryonated eggs were collected.The bacteria were isolated from the liver of chick embryos and the morphology of isolates was observed.The DNA of the isolates was extracted and 16S rDNA gene was amplified.These isolates were identified by 16S rDNA alignment.The results showed that there were cracks or fecal contaminants in the appearance of embryonated egg,and jelly samples under the skin,the liver khaki,and the yolk malabsorption were found in chick embryos.A total of 86 strains were isolated and identified from embryonated eggs.The contamination rate was 59.3%(54/91)and the mixed contamination rate was42.6%(23/54).11 species of bacteria were identified,of which Escherichia coli proportion was 37.2%(32/86),Enterococcus faecalis proportion was 26.7%(23/86),Shigella proportion was 9.3%(8/86),Enterobacter cloacae proportion was 5.8%(5/86),Bordetella proportion was 4.7%(4/86),Salmonella proportion was 4.7%(4/86),Staphylococcus aureus proportion was 3.5%(3/86),Acinetobacter baumannii proportion was 3.5%.(3/86),Pseudomonas psychrotolerans proportion was 2.3%(2/86),Massilia alkalitolerans proportion was 1.2%(1/86)and Pseudomonas aeruginosa proportion was 1.2%(1/86).These information suggest that the contamination of embryonated eggs involves a variety of pathogenic bacteria,the main contaminating bacteria are Escherichia coli and Enterococcus faecalis,which provides a basis for the safe production and monitoring of embryonated egg products.2.In order to establish a method for rapid detection of Escherichia coli in chicken embryonated eggs,a pair of specific primers were designed according to the Escherichia coli fimC gene,and a real-time quantitative PCR method was established.The chicken embryonated eggs sample test has no cross-reaction with Salmonella,Shigella,Proteus mirabilis,Pseudomonas aeruginosa,Enterococcus faecalis,Enterococcus faecium,Staphylococcus aureus and other pathogenic bacteria,and has strong specificity;The minimum detection limit for Escherichia coli is 10~1 CFU/mL,which is 100 times more sensitive than conventional PCR method and has good sensitivity.The coefficients of variation were less than 2%in the repeatability test,indicating that the repeatability was good.The method was stable and could be used for the detection of Escherichia coli in chicken embryonated egg products.3.In order to establish a method for rapid detection of Enterococcus faecalis in chicken embryonated eggs,a pair of specific primers were designed according to the Enterococcus faecalis sodA gene,and a real-time quantitative PCR method was established.The chicken embryonated eggs sample test has no cross-reaction with Enterococcus faecium,Staphylococcus aureus,Escherichia coli,Salmonella,Shigella,Proteus mirabilis,Pseudomonas aeruginosa and other pathogenic bacteria,and has strong specificity;The minimum detection limit for Enterococcus faecalis is 10~1CFU/mL,which is 100 times more sensitive than conventional PCR method and has good sensitivity.The coefficients of variation were less than 2%in the repeatability test,indicating that the repeatability was good.The method was stable and could be used for the detection of Enterococcus faecalis in chicken embryonated egg products.4.A duplex quantitative PCR method was established for the Escherichia coli and the Enterococcus faecalis.The results showed that the Tm values of Escherichia coli products and Enterococcus faecalis products were 85.6-86?and 83.9-84?respectively,and there was no cross-reaction with other bacteria.The coefficient of variation in the repeatability test of Escherichia coli and Enterococcus faecalis was less than 2%,and the method had good repeatability.The detection limits for Escherichia coli and Enterococcus faecalis were 10~1 CFU/mL.It is suggested that the duplex quantitative PCR method is sensitive,specific,stable and reliable.Simultaneous detection of Escherichia coli and Enterococcus faecalis in one reaction can be used for the detection of chicken embryonated egg products.