| Objective1. A rapid detection method of multiplex PCR was established to detect 10 virulence genes of 80 B. cereus strains collected in Fuzhou, as well as quickly identify various strains of Bacillus.2. A method of pulsed field gel electrophoresis(PFGE) was established to assay the molecular typing and homology of 52 B. cereus strains. Foods collected from different areas at different times are traced to provide scientific data for diseases control.Methods1. 300 infant food samples, including rice powder and milk powder, were collected and isolated B. cereus strains by bacteria increasing, isolating culture and biochemical test.2. Based on hbl A, hbl C, nhe A, nhe B, cyt K, cer,ent FM and bce T genes of B.cereus, cry gene of B.thuringiensis and pag gene of B.anthracis, primers were designed and reaction conditions were optimized to rapidly identify B. cereus and B.thuringiensis as well as establish a multiplex PCR detection method for virulence genes of B. cereus.3. Virulence genes of 80 B. cereus collected in Fuzhou were detected by multiplex PCR and the results were confirmed and compared by single-target PCR and national standard with strains of 20 Listeria, 10 Staphylococcus aureus and 10 Salmonella as negative controls.4. The strains of B.cereus were hydrolyzed and incubated in lysozyme solution at 37 ?for 6h, and then digested with proteinase K at 54? overnight. PFGE method was applied in the genetic typing study on B. cereus by using Not I, Sba I and Smart I with different enzyme concentration and hydrolysis time.5.52 B.cereus strains isolated from infant food samples were hydrolyzed with endonuclease Not I and typed by PFGE. The results of genetic typing were analyzed by Bionumerics 5.10.Results1. 80 isolates of of B.cereus were detected by multiplex PCR method in the 300 infant foods samples, The positive rate was 26.66%(80/300) and the colony quantitive range was 10-800 CFU/g.2.10 virulence genes of B.cereus strains were detected by single PCR, The positive rates of hemolysin hbl A, hbl C and nonhemolytic nhe A,nhe B was 57.5%, 25% and75.0%, 80.0% respectively, and cyt K gene is 37.5%, ent FM gene is 28.7% and bce T gene 18.7%. The detection rates of cer gene were 97.5% and 100% in B.cereus and B.thuringiensis,and cry gene were 0.0% and 100% respectively.Pag gene were not detected both in B.cereus and B.thuringiensis.These results were equate to those of Mousumi banerjee.3.10 virulence genes of B.cereus strains were also detected by multiplex PCR, The positive rates of hemolysin hbl A, hbl C and nonhemolytic nhe A,nhe B was 56.3%,25.0% and 73.80%, 77.5% respectively, and cyt K gene was 36.2%, ent FM gene was27.5% and bce T gene 20.0%. The detection rates of cer gene were 95.0% and 100% in B.cereus and B.thuringiensis,and cry gene were 0.0% and 100% respectively.Pag gene were not detected both in B.cereus and B.thuringiensis. The results of the two methods of single PCR and multiplex PCR had a high coincidence rate. The electrophoresis strips of the three pairs of primes were clearly distinguishable. The sensitivity of multiplex PCR method to virulence genes was 102CFU/m L.4. No cross-reactive results were observed with the control groups of 20 Listeria strains,10 Staphylococcus aureus strains and 10 Salmonella strains. It indicated that the designed primers were highly specific for detecting virulence genes of B.cereus.5. The reaction conditions of PFGE typing were as follows: B. cereus was hydrolyzed and incubated with lysozyme at 37 ? for 6 h and digested with endonuclease Not I at the concentration of 10U/?l for 3 h.6. The results of homology analysis of the 52 strains of B. Cereus from the lab of Fuzhou Center for Disease Control and Prevention were as follows: based on 100%similarity, the 52 strains could be divided into 47 PFGE types without domain type.Only 5 strains collected from different manufacturers at different times showed identical in PFGE type.Conclusion1. The multiplex PCR method in this test was proven to be highly sensitive, specific as well as efficient for rapid detection of B. cereus. The results were quite satisfied, which was beneficial for rapid and accurate report and had important practical significance.2. The optimized reaction conditions of PFGE method was established to evaluate genetic typing of B. cereus in the study. The method was highly repeatable, sensitive and could meet the typing requirements. Foods collected from different areas at different times could be traced to provide effective means for tracing food-borne diseases and nosocomial infection caused by B.cereus as well as reducing lost in food industry caused by B. Cereus and controlling diseases. |
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