Rapid Detection Of Salmonella In Dairy Products By IMBs-qPCR And Multiple PCR

In recent years,food safety problems occur frequently,which makes consumers pay more and more attention to the safety of dairy products.The main problems affecting the food safety of dairy products are pathogenic microorganisms,pesticide residues,antibiotics and heavy metals,among which the most important pathogenic microorganisms are dairy products.Detection of pathogenic microorganisms is traditional method training methods,including:pre enrichment?18-24 h?and selective enrichment?24-48 h?,biochemical reaction,microscope observation and determination of serum type,but this conventional method is not suitable for rapid detection.To establish a rapid method for detection of pathogenic microorganisms and to improve the detection speed and efficiency is of great significance in food safety.Immunomagnetic separation technology is on the surface of microspheres added metal molecule,then antibody plus microsphere surface,which are combined with the target antigen,under the action of magnetic force set down,and thereby to protein impurities were separated and enriched and separated,so as to achieve the purpose of purification of the target protein.Through bioinformatics analysis,the PagC gene was found on GeneBank and specific primers were designed to amplify PagC gene.Pag C protein was expressed and purified by prokaryotic expression.Polyclonal antibodies were prepared by purified PagC protein,and antibodies were conjugated to magnetic beads by the special coupling reaction of magnetic beads.The results showed that the best amount of immunomagnetic beads was 116 g mg antibody per bead.0.1 mg magnetic beads can capture Salmonella concentrations of 10¹¹0⁴CFU/mL.The detection of Salmonella was carried out by combined immunomagnetic bead separation and fluorescent quantitative PCR,and the detection limit of Salmonella was18CFU/mL.The detection time of this method is less than 10 h.Compared with the national standard method,the detection time is shortened obviously.Multiple PCR detection of food pathogenic microbes has a high sensitivity and can reduce the error in detection.In this experiment,we first determined the conditions of multiplex PCR for detection of Salmonella.The best primers were invA 0.3,L,fliC 0.2,L and sdfI 0.3 L,and the best Mg²⁺,dNTPs and Taq DNA enzymes were 4,5,4 and 0.4,respectively.In this experiment,the multiple PCR was used to reduce Salmonella in dairy products.The results showed that the multiple PCR detection of Salmonella was highly specific.Compared with the national standard detection method,the results showed that the detection time of IMBs-qPCR was shorter than that of the national standard,and the pathogenic bacteria could be rapidly detected in the food detection.Multiplex PCR detection of high sensitivity and specificity in the food testing process can avoid the phenomenon of misjudgment.Therefore,in the actual testing process should be used in both ways,you can quickly detect food-borne pathogens at the same time avoid missing and misjudged.