| Food-borne pathogens have been harming human health by affecting food safety.In recent years,food safety incidents have repeatedly appeared,which not only threatens people's lives and work to a certain extent,but also has a close relationship with the future development of the country.Bacterial food poisoning is the main cause of food-borne diseases.Campylobacter jejuni(C.jejuni)is the most serious cause of food-borne diseases.There are many difficulties in the detection of these pathogenic bacteria,which can not be fully applied to today's detection research.Therefore,it is urgent to establish a new detection method for C.jejuni.Common PCR method can achieve exponential growth of target in a very short time,and improve sensitivity to a large extent;and immunochromatography method is also easy to produce,low cost and can achieve sensitive and rapid detection,which is widely used in the detection of various foods.This study is a new method which combines common PCR and immunochromatography technology.It has successfully completed the detection of C.jejuni with low detection limit,high specificity and simple and rapid.(1)Establishment of a single PCR-based method for simultaneous detection of C.jejuni virulence gene.In this chapter,the specific primers of C.jejuni conservative sequence mapA and virulence gene cdtB were designed by consulting data,and then amplified by PCR using C.jejuni template DNA.Finally,the amplified products were characterized by agarose gel electrophoresis,and a new high sensitivity and high specificity method for detecting C.jejuni was established.During the experiment,the extraction conditions of template DNA in the preparation of samples for PCR amplification,the annealing temperature,primer concentration and magnesium ion concentration in the process of PCR amplification were optimized.The final detection results showed that the detection limit reached by this method was 1.1×10~2 CFU/mL,and the results were very stable after repeated verification.(2)Preparation of C.jejuni virulence gene test strip based on colloidal gold labeling and its application in milk samples.In this chapter,based on the previous chapter of PCR,we modified digoxin,Biotin and digoxin and FITC with upstream and downstream primers of mapA and cdtB,respectively.Two double-stranded nucleic acid molecules with digoxin,Biotin and digoxin and FITC at both ends were obtained.The target was captured on the test strip by sandwich method.Finally,a rapid C.jejuni detection method was established by observing the appearance of the test strip.Method.In this experiment,the optimum amount of gold label antibody,the optimum blocking agent and the optimum formula of heavy suspension were explored.The 11 CFU/mL detection of C.jejuni was completed.Compared with the final experimental results in the previous chapter,the sensitivity was increased by about 10 times.And through the identification of adulterated milk samples,the final detection results show that the C.jejuni detection method established in this experiment has important application prospects in food safety control and detection.(3)Preparation of C.jejuni virulence gene test strip based on quantum dot labeling and its application in food detection.On the basis of the previous chapter,the labels are replaced by quantum dots of various colours.The optimum antibody quantity,blocking solution,recipe of resuspension and pH of diluent are optimized.A new method for detecting fluorescent strips of colloidal gold with different colours of quantum dots is established.The aim is to identify C.jejuni and its virulence by comparing different colors.The results showed that C.jejuni's marker factor mapA and its virulence identification factor cdtB could detect 11 CFU/mL with good specificity,which could be applied in food monitoring. |
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