Study On Rapid Detection System Of Salmonella And Its Multidrug-resistant Analysis

The thesis has established the rapid detection system of salmonella firstly,including gold-immunochromatography assay, dot immunogold filtration assay,universal PCR, real-time fluorescence quantitative PCR. Aseries of parametersare optimized, and they form ripe reagent kits. The reagent kits have beenapplied to investigate on salmonella contamin- ation of the products of Iovestockand poultry in Xiamen area. They shall short the detection period from 4d～5d to2d. Moreover, the experiment of antimicrobial susceptibility and multi- drugresistant analysis was done to know actual drug resistance on Salmonellastrains isolated from Xiamen area. Thirdly, multiplex polymerase chain reactionis developed to detect two kinds of resistant genes of tetracycline (tetB and tetC).The research abstract was summarized as follows:1. Application andresearch of gold-immunochromatography assayof Salmonella. To establish a more rapid and simple method of detectingsalmonella, the gold immunochromatography assay(GICA) was developed bycoating rabbit anti-salmonella IgG-2 on nitrocellulose(NC) membrane, sprayingsheep anti-rabbit IgG on control line, labeling rabbit anti-salmonella IgG-1 withcolloidal gold. If detected sample contains salmonella, the detection lineappears red line. The whole test procedure could be finished in only10～15minutes. The method has a better specificity and no cross-reaction withother enterobacteria such as Escherichia coil,proteus mirabilis,Citrobacterfreundii,Enterobacter cloacae, its sensitivity is 2.3×10~5 cfu/ml. It can be Storedat 4℃or 37℃for 9 months and maintain good detecting result. So the strip hasa simple,rapid,high sensitive,strongly specific characteristics, doesn't needspecific instruments and facilities, and its result is easily observed anddetermined. It is good worthy to use it widely, especially in small diagnosticlaboratories.2. Application and research of dot immunogold filtration assay forthe detection of Salmonella. To establish a more rapid and simple method ofdetecting salmonella, the dot immunogold filtration assay (DIGFA) wasdeveloped by coating clinic sample on nitrocellulose(NC) membrane, sprayingSPA on control dot, labeling rabbit anti-salmonella IgG-1 with colloidal gold. If detected sample contains salmonella, the detection dot appears red spot. Thewhole test procedure could be finished in only 10～15minutes. The method has abetter specificity and no cross-reaction with other enterobacteria such asEscherichia coil,proteus mirabilis,Citrobacter freundii,Enterobacter cloacae,itssensitivity is 4.5×10~6 cfu/ml. It can be Stored at 4℃for 12 months and accordwith native criteria. So the DIGFA has a simple,rapid,high sensitive,stronglyspecific characteristics, doesn't need specific instruments and facilities, and itsresult is easily observed and determined. It is good worthy to use it widely,especially in small diagnostic laboratories.3. Research and application on rapid detection kit of salmonella bypolymerase chain reaction (PCR). This study established PCR to detectSalmonella species according to fimY gene. The specific primers to the fimYgene were used to amplify standard bacteria of A-F salmonella,60 salmonellaseparate strains and 11 non-salmonella species by PCR. Experiment resultsindicated that only Salmonella species generated the specific 526bp DNAfragments on the 2.0% agarose gel and non- salmonella species controls gavenegative results, demonstrating that the primers should be specific forSalmonella genus. The PCR was able to detect as little as 93cfu Salmonella andpre-cultured(BP) or cultured(MM) bacteria liquid of the sample obtaining 3cfuSalmonella. This indicates established PCR method is high sensitive andstrongly specific. The nucleic acid is purified by five methods, including addingbacteria,hot cleavage,freezing-thaw again and again,CTAB and columnreagent kit, the CTAB method IS best, but testing procedure is multifarious.Adding bacteria and hot cleavage can procure the same result as columnreagent kit and have many merits including simple,rapid,economical and isworthy to generalization and application. This research has established asimple,sensitive and specific method for the detection of salmonella genus.4. Application and research on the rapid detection kit by real-timefluorescence quantitative PCR in Salmonella. Salmonella is a majorzoonose bacteria and the most common food poisoning factor in bacterial foodpoisoning. It is very important meaning to establishing a simple,sense,accuratemethod for rapid detection in order to preclude food poisoning caused bysalmonella. The Taqman probe and primers were designed and sythesizedaccording to the conserved gene fimY of salmonella available in GenBank, andthen reaction parameters were optimized to develop a real time Taqmanquantitative PCR detection kit. The developed quantitative PCR kit could detect4.5cfu/25uL (a reaction system), its sensitivity was 100 times higher than thatof the routine PCR, and it could be stored over nine months in -20℃. To detectsome clinic samples, the developed kit has many advantages such as simple, sense,specific,accurate,good repeated and stable method for rapid detectionin salmonella and is applied to the sample detection for a large number.5. Application on seria of rapid detection methods of salmonella.The investigation on salmonella contamination of the products of Iovestock andpoultry in Xiamen area was carried out with conventional technique and colloidalgold strip. From 2004/5 to 2006/10, We surveyed the products of Iovestock andpoultry, fishmeal, feedingstuff, water and feces in hoggery. 81 strains ofsalmonella bacteria were detected from 4705 samples, the positive rate was1.72%, and they were validated by universal PCR technique and real-timefluorescent quantitative PCR technique. Some strains were not reported inChina such as S.Namibia(serum type, 6,7: c,enx) detected in feedingstuff fortortoise. The same results were obtained with four detection techniques. As awhole, the products are better in Xiamen supermarket and have contaminatedrate of 0.43%(9/2350), but contamination status is more serious in hoggery,porcine products and live poultry in small bazaar, and Gaoqi slaughterhouse,their contaminated rates were 26.7%(4/15), 9.33%(14/150), 2.67%(8/300),10%(40/400) separately. The management is enhanced in livestock breedingand current tache, specifically in disinfection and cleanness.6. Development and application of detection of two kinds of resistantgenes of tetracycline (tetB and tetC) by multiplex polymerase chainreaction. A multiplex PCR method was developed to simultaneously amplifytwo genes, tetracycline B and tetracycline C. Thirty-five salmonella isolates in ourcollection gave 100% (35/35)tetC and 22.86%(8/35)tetB positive reactions to thisPCR assay by amplifying 418bp(tetC)and 659bp (tetB) PCR products.Compared to the results of the drug susceptibility test, PCR showed65.7%(23/35)concordance in positive rate and 100% concordance to salmonellasimultaneously containing tetB and tetC genes. The sequencing showed tetBgenes from five isolated salmonella strains were 99.7% of homology with thatfrom pRT11 and 100% of homology one another; tetC genes from fourteenisolated salmonella strains were 100% of homology with that from pBR322. Theresults showed that antibiotic resistance genes were commonly present in theisolated strains. The established multiplex PCR technique has many advantages(including simple,rapid,save money and time) and is specially adapted fordetecting a lot of samples. It could also be a useful method to carry out molecularepidemiology surveillance of multiplex tetracycline resistance genes.7. The antimicrobial susceptibility test on Salmonella strains isolatedfrom Xiamen area and multi-drug resistant analysis, The Salmonellastrains isolated from Xiamen area were studied for antimicrobial susceptibilitytest and multi-drug resistance. 59 strains of Salmonella were tested using 24 kinds of antibiotic paper. The rates of these strains resisting to antibiotic showedan increase -ing tendency, especially to Tetracycline, Doxycycline, Ampicillin,Penicillin, Erythromycin, Rifampicin, Midecamycin, Novobiocin, Furazolidoneand Chloramphenicol, and even some strains were drug resistence to higheffective antibiotics such as Streptomycin, Gentamycin, Kanamycin, Cefamezin,Cephradine and Ciprofloxacin. The strains having the capacity of drugresistance to three～ten, twelve, fourteen and fifteen types antibiotic are 2, 5, 6,13, 11,7, 3, 2, 1, 5 and 4, and their percentages are 3.4, 8.5, 10.2, 22.0, 18.6,11.9, 5.1,3.4, 1.7, 8.5 and 6.8. Moreover, 51 strains of Salmonella are analyzedfor drug resistant degree of 5 kinds of antibiotics, the results show over 16ug/mldrug resistances have 35 strains (68.6%,35/51)in tetracycline,23 strains(45.1%,23/51) in streptOmycin, 14 strains(27.5%, 14/51)in Ampicillin,31 strains(60.8%,31/51) in Chloramphenicol and over 1600ug/ml Sulfame resistance has51(100%, 51/51). Therefore, establishing surveillance system for drug resistanceof Salmonella and searching for methods curing plasmids are an urgent problemof prevention. This indicates drug resistance in bacteria has been more seriousand remind human to use drug accurately.