| Salmonella is an important food-borne pathogen.It can infect humans and cause fever,vomiting,diarrhea and other symptoms after human food poisoning,and even death in severe cases.Animal-derived food is one of the important ways of Salmonella transmission.To establish a rapid and efficient Salmonella detection method can more effectively monitor the Salmonella contamination,which is of great significance in food safety.This study mainly included two aspects:First,the immunomagnetic beads combined with polymerase chain reaction(IMBs-PCR)method was optimized in the laboratory.Second,the contamination and drug resistance of Salmonella spp.from cattle and sheep in the vicinity of Urumqi were investigated,and the virulence genes of the isolates were detected.(1)Through the orthogonal experiment,we could find that the effects of the amount of immunomagnetic beads,mixing time and bacterial solution concentration on the capture rate of immunomagnetic beads were in the order of IMBs amount>bacterial solution concentration>mixing time.The optimal amount of IMBs was 20μL/m L,the optimal incubation time was 10 min,and the optimal sample concentration was 10~3 CFU/m L.The specificity of the immunomagnetic beads was detected,and the results showed that the capture rates of Salmonella immunomagnetic beads to E.coli and Proteus mirabilis were low,and the average capture rate to Salmonella was high.The detection limit results show that Salmonella immunomagnetic beads can successfully capture Salmonella at a concentration of 3 CFU/m L.A total of 65 milk samples were simultaneously detected by IMBs-PCR and conventional methods,and eight positive samples were detected by both methods respectively.The detection time by IMBs-PCR was about 8 h.(2)One hundred and eighteen Salmonella strains were identified from 1 529 samples(1 282of bovine origin and 247 of sheep origin,including 908 fecal samples,65 milk samples,16meat samples,62 feed samples and 478 swab samples).The overall isolation rate was 7.72%,among which the isolation rate of Salmonella spp.from cattle was 4.06%(52/1 282),and that of Salmonella spp.from sheep was 26.72%(66/247).The coding conditions of 18 virulence genes are detected by a PCR method,sip B(94.92%),spi A(92.37%),ara B(92.37%),ssa Q(92.37%),inv J(91.53%),sop B(88.98%),mog A(88.14%),sif A(84.75%),mgt C(64.41%),sse L(61.86%)on the Island of Toxicosis.The detection rates of lpf C(88.14%)and bcf(62.71%)on fimbriae and stn(92.37%)of enterotoxin were higher than those of spv A(13.56%),spv B(1.69%),spv C(11.86%),spv D(0.85%)and spv R(30.51%)on virulent plasmid.spv B,spv C and spv D on the virulent plasmid were not detected in Salmonella of sheep origin,and the detection rates of virulent genes of virulent island,fimbriae and enterotoxin in Salmonella of sheep origin were higher than that of Salmonella of cattle origin.The drug resistance rates of 118 Salmonella strains to 13 antimicrobial agents were tetracycline(77.97%),ampicillin(76.27%),amoxicillin-clavulan ate(72.88%),polymyxin E(62.71%),ceftiofur(59.32%),cefoxitin sodium(52.54%),ceftriaxo nesodium(48.31%),kanamycin(41.53%),and gentamicin(34.75%).Enrofloxacin(33.90%),azithromycin(16.95%),chloramphenicol(6.78%),and ciprofloxacin(0.85%).In addition,the isolate was completely sensitive to sulfamethoxazole,and the resistance rate of Salmonella spp.of sheep origin was higher than that of Salmonella spp.of cattle origin.There were 100 multi-drug resistant strains,mainly distributed among 8 resistance(13.56%),6 resistance(12.71%),5resistance(11.86%),9 resistance(11.86%)and 3 resistance(11.02%).Salmonella spp.of sheep origin showed serious multi-drug resistance,which was higher than the multi-drug resistance rate of Salmonella spp.of cattle origin.The IMBs-PCR method was suitable for the detection of Salmonella in milk due to its good specificity and short detection time.The pollution level of Salmonella of sheep origin around Urumqi was higher than that of Salmonella of cattle origin,and the pollution situation was serious.Salmonella of cattle and sheep origin carry a large number of virulent genes and have high resistance to commonly used antibiotics and multi-drug resistance is serious. |
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