| Nowadays,that the outbreak mode of disease caused by microbial contamination has changed from small centralized outbreaks in local areas to large outbreaks consisting of scattered cases in the whole society.In order to effectively identify large outbreaks,a series of traceability analysis methods have emerged to identify and track the transmission pathways of food microorganisms.In order to meet the current demand for rapid and high-throughput detection,molecular biological methods have been widely used in the detection of food microorganisms,however,the relevant reference substances are scarce.According to the different management agencies in China,the reference material(RM)is distinguished as standard substances(GBW)and standard samples(GSB),of which the certified reference material(CRM)refers specifically to the reference material with a certificate.Standard samples are the yardstick for measuring the quality control work of testing laboratories,and they are widely used in laboratories at all levels of health prevention,port supervision,food safety production and supervision departments,as well as third-party testing laboratories.The development of ready-to-use standard samples for food-borne microbiological testing can help solve the problem of insufficient reference materials for domestic food microbiological testing and enable the promotion and application of standard samples with independent intellectual property rights in the field of food microbiological testing,so as to get rid of the reliance on foreign standard samples for food microbiological testing.In response to the lack of plasmid of certified reference material suitable for detection of Clostridium botulinum and Vibrio vulnificus and the detection method of Salmonella stanley in China,certified reference material of Clostridium botulinum plasmids containing type A,type B,type E and type F toxin genes and certified reference material of Vibrio vulnificus plasmid of vvh A,vcg C/E,16 S r RNA A/B,Bt2/Ser E were developed in this thesis.Dual priming oligonucleotidepolymerase chain reaction(DPO-PCR)systems were established for detecting Clostridium botulinum and Salmonella stanley,respectively.Certified reference material of four botulinus toxin gene plasmids,four Vibrio vulnificus plasmids of identify and genotyping,and genome of Salmonella stanley were applied to food detection.Specific research contents are as follows:(1)The recombinant plasmids containing Clostridium botulinum toxin genes were constructed,and 450 tubes of freeze-dried powder of plasmids were prepared after sequencing verification.Referring to the requirements of series of standards of GB/T 15000.3-2008 "Guidelines for working with standard samples",12 tubes were randomly selected according to the random function,and the certified reference material of botulinum toxin gene plasmids were subjected to PCR qualitative detection referring to GB 4789.12-2016 "National food safety standard for food microbiological test of Clostridium botulinum and botulinum toxin examination".Meanwhile,the concentration of plasmid samples was determined by UV spectrophotometer method,and the statistical analysis of plasmid content results was performed by ANOVA.The results showed that using the certified reference material of 4 kinds of Clostridium botulinum toxin gene plasmids as templates,the target gene fragments with correct size and single amplification band could be amplified.Agarose electrophoresis analysis showed that the plasmid samples had intact bands without degradation.Quantitative statistical analysis of the plasmid samples showed that F values were all lower than F critical value,indicating that there was no significant difference between the samples and the uniformity was good,which met the expected target.Since temperature is the main factor affecting the stability of the certified reference material during transportation,the short-term stability analysis of the samples was carried out under the transport temperature conditions of 4°C,37°C and 45°C.Three tubes of samples were taken for detection and analysis on the 1st,3rd,5th,7th,9th,11 th and 14 th day,respectively.At each sampling time point,using plasmid of certified reference material as template,the target gene fragment with correct size and single amplification band could be obtained.Agarose electrophoresis analysis showed that the plasmid samples had intact bands without degradation.According to GB/T15000.3-2008/ISO Guide 35:2006 “Working Guidelines for Standard Samples(3)General Principles and Statistical Methods for Standard Sample Value”,linear fitting cooperation was adopted as the basic model for stability research.The t-test analysis results showed that the slope was not significant,indicating that no instability was observed in plasmid samples.These results indicated that the plasmid samples of Clostridium botulinum toxin gene could be stably stored at4°C and 37°C for 14 days.The samples remained stable at 45°C for 9 days.In addition to transport stability,the long-term stability(storage stability)of the plasmid samples was also analyzed.Samples stored at-20°C were analyzed at the 1st,2nd,4th,6th,8th,10 th,and 12 th month,respectively.The results showed that the target gene was detected in the plasmid samples at each time point,and the electrophoresis bands were clear.The electrophoresis bands of the plasmid samples were complete,and no instability was observed by quantitative t-test,indicating that the samples could be stably stored at-20°C for at least 12 months.The plasmid samples were determined jointly by 8 units,such as the National Center for Food Safety Risk Assessment,Shandong Institute of Food and Drug Inspection,Dandong Customs Comprehensive Technical Service Center certified by China National Accreditation Service for Conformity Assessment(CNAS)and other institutions with relevant qualifications.The results from the 8 units showed that the 4 certified reference materials were consistent with expectations.In conclusion,the certified reference material of Clostridium botulinum toxin gene plasmid conforming to commercial requirements was constructed.(2)The certified reference materials of plasmids containing the genes from Vibrio vulnificus were prepared according to the method of the certified reference material of the toxin gene plasmid of Clostridium botulinum.Firstly,four recombinant plasmids containing virulence gene vvh A and typing genes of vcg C/E,16 S r RNA A/B,Bt2/Ser E were constructed respectively,and 450 tubes of freeze-dried powder of plasmids were prepared after sequencing verification.The certified reference materials of plasmid were qualitatively detected by PCR according to GB 4789.44-2020 "National standard for food safety microbiology test of Vibrio vulnificus examination".At the same time,UV spectrophotometer was used to determine the concentration of plasmid samples for quantitative analysis.The uniformity test results showed that there was no significant difference between the samples,and the uniformity was good and in line with the expected target.The results of short-term stability test showed that plasmid samples could be stably stored for 14 days at 4°C and 37°C.Long-term stability test showed that plasmid samples could be stably stored at-20°C for at least 12 months.The above results indicated that the uniformity and stability of certified reference material of plasmids met the requirements of national certified reference material,which provided a reliable reference material for rapid and high-throughput qualitative identification and typing analysis of Vibrio vulnificus.(3)A quadruple DPO-PCR system was established for the detection of Salmonella stanley.Compared with the traditional PCR method,the method had a good amplification effect at 49°C-58.5°C with clear bands,good specificity,and a sensitivity of 0.7 ng/μL.Two positive samples were identified from 95 food samples by this technique,which were verified by traditional culture method,indicating that this technique is accurate and reliable.Therefore,a sensitive and rapid Salmonella stanley detection system was established.(4)A quadruple DPO-PCR assay system was established to detect Clostridium botulinum.The results showed that the DPO-PCR system using the certified reference materials of Clostridium botulinum toxin gene plasmids as template could amplify four target bands under the annealing temperature of 53.6°C-62°C,with a sensitivity of 0.002 ng/μL and good specificity at a time.In the applicability test of 15 food samples,the detection rate was 100%,indicating that the certified reference material of the toxin gene plasmid of Clostridium botulinum was practical and effective,and the established quadruple DPO-PCR detection system provides a new scheme for the rapid detection of Clostridium botulinum.(5)In order to test the application of Vibrio vulnificus plasmid of certified reference material in food detection,four Vibrio vulnificus plasmids of certified reference material were used as positive controls for testing 10 seafood samples,including fish and shellfish,according to the test method of GB 4789.44-2020 "National standards for food safety microbiology test of Vibrio vulnificus examination".The results showed that one positive sample was detected among all the samples,which was confirmed to be Vibrio vulnificus by traditional culture method.Therefore,it is proved that the certified reference material of Vibrio vulnificus plasmid developed in this study has good commercial application potential,which lays an important foundation for its further popularization and application.In this study,certified reference material of plasmid for rapid detection of Clostridium botulinum and Vibrio vulnificus in food were developed,and their accuracy and reliability were verified in food detection,which laid a strong material basis and technical support for the implementation of GB4789.The establishment of plasmid certified reference material eliminates the culturing steps of positive control pathogens,which is not only cost saving,but also ensures the personal safety of inspectors in food inspection institutions and food safety supervision departments.In addition,the DPO-PCR systems of Clostridium botulinum and Salmonella stanley were constructed,which provided a new idea and scheme for the rapid detection of these two microbes,eliminating the tedious operation of multi-gene detection,greatly shortening the detection time and improving the detection efficiency.In summary,the research results of this thesis provide solid and strong support for the efficient and rapid detection of Clostridium botulinum,Vibrio vulnificus and Salmonella stanley in food test,and also help to improve the level of rapid detection of foodborne pathogens in China. |
PDF Full Text Request