| Objective: To develop a rapid and accurate protocol for the detection of Salmonella, which is important pathogenic agents for both human beings and animals.Methods: (1) Gold immunochromatography assay(GICA): At the beginning , the pure antiserum was obtained from rabbits immunized by several successive injections of cell suspensions of Salmonella and polyclonal antibody was extracted from the serum using Protein A Sepharose TMCL-4B. In the second place,the colloidal gold of 34nm for labeling Polyclonal antibody of the bacteria was produced through reducing or deoxygenating the gold chloride with trisodium citrate.Then the Polyclonal antibody was mixed with the 34nm colloidal gold with a concentration of 15ug /ml to produce gold-labeled Polyclonal antibody mixture.After that,a test strip for Salmonella was assembled with nitrocellulose membrane,the purified antiserum,the gold-labeled polyclonal antibody,sheep anti-rabbit immunoglobulinG(Purchased) and glass fibrous membrane (Purchased).Finally,the test strip was examined for its spccificity and sensitivity.The life of the test strip was also investigated under different storage conditions.(2) Fluorescencer quantitive polymerase chain reaction(FQ-PCR): At first,the Taqman probe and primers were designed and sythesized according to the conserved gene fimY of salmonella available in GenBank.Second,the reaction parameters were optimized to develop FQ-PCR.Finally,the sensitivity, the specificity and the reproducibility was evaluated.(3) GICA and FQ-PCR were used to detect Salmonella in feed, dung of swine,meat and so on, depending on the National Standard(GB) method to verify the results.Results: (1) GICA has a better specificity and no cross-reaction with other enterobacteria such as Escherichia coli,Proteus mirabilis,Enterobacter cloacae. Its sensitivity is 2．3×10~5 cfu /ml. It can be Stored at 4℃or 37℃for 9 months and maintain good detecting result. In addition, the whole test procedure could be finished in only 10~15minutes.(2) FQ-PCR could detect 4．5 cfu per reaction, and its sensitivity was 100 times higher than that of the routine PCR,otherwise, the detection results of different strains of non-Salmonella and Salmonella showed high specificity of this assay and the reproducibility is good,too.(3) The resuluts of detecting samples showed that the sensitivity and specificity of combination use of two methods (GICA and FQ-PCR) was 100%, compared with the results of National Standard method. It also indicated that this combined methods is more quick and simple than National standard method.Conclusion:Based on all the results,an optimized protocol for the detection of Salmonella was developed,that is,to use GICA to screen the large number of sarnples first,and then to use FQ-PCR to test the GICA positive samples.The final step is,if needed, to use National Standard method to determine the serotypes of Salmonella.This combined method was highly specific sensitive and very quick.lt provides an efficient tool to handle many samples in animal quarantine,food and feed hygiene,diseases prevention.This procedure ensures the accuracy and reliability of detecting results and saves lots of material and manpower,which would create great social and economic interest.
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