摘要
细菌性食物中毒占我国食物中毒总数的首位,动物性食品是引起细菌性食物中毒的主要原因。目前对食品中致病菌检测的方法以微生物常规培养法为主,需要3~7天,操作繁琐,所用试剂复杂多样,费用很高,不能满足实际商品流通需要。PCR(聚合酶链式反应)技术具有特异性强、灵敏度高、操作简单、快速的特点,使用PCR技术检测食品中致病菌的研究方兴未艾。大肠埃希氏菌O157:H7、单核细胞增生李斯特氏菌、小肠结肠炎耶尔森氏菌和荧光假单胞菌是污染动物性食品的四种重要的致病菌。本研究结合通用前增菌技术研究,建立了一种多重PCR检测新方法,可以在LB培养基通用增菌后的6h内一步同时检测食品中污染的这四种致病菌。该方法具有特异性良好、简便快速、经济实用的优点。研究结果如下:
(1)对大肠埃希氏菌O157:H7、副溶血性弧菌、伤寒沙门氏菌、宋氏志贺氏菌、金黄色葡萄球菌、创伤弧菌、单增李氏菌、小肠结肠炎耶尔森氏菌、荧光假单胞菌等九种食源性致病菌进行通用前增菌方法研究,实验表明,在LB肉汤培养基36℃培养18 h,增菌效果最好,且增菌数量级均衡一致,达108 cfu/mL。
(2) LB、NB和UPB培养基中30℃、33℃、36℃培养12h,大肠埃希氏菌O157:H7、副溶血性弧菌、伤寒沙门氏菌、宋氏志贺氏菌、金黄色葡萄球菌、创伤弧菌、单增李氏菌、小肠结肠炎耶尔森氏菌、荧光假单胞菌等九种菌增菌数量级达到106以上,也能够满足PCR等检测方法的需要。但这几种培养基在36℃的培养时间应选在12~18 h左右,不宜超过18 h。
(3)根据大肠埃希氏菌O157:H7的eaeA基因,单核细胞增生李斯特氏菌的hly基因,小肠结肠炎耶尔森氏菌的ail基因,荧光假单胞菌的htpX基因设计了4对引物。实验证实具有良好的特异性。
(4)建立了大肠埃希氏菌O157:H7、单核细胞增生李斯特氏菌、小肠结肠炎耶尔森氏菌和荧光假单胞菌的单重和多重PCR检测方法,并确定了多重PCR体系和热循环参数。经实验证实了所建立的方法特异性很强。
Bacterial food poisoning accounted for the top of the total number of China's food poisoning. Animal food is the main reason for food poisoning caused by bacterium. At present, the main method of pathogens detection in food is conventional culture-based microbiological, which needs 3 to 7 days and not easy to operate, and the reagents used are complex ,diverse, and high cost . That’s the reason for the methods can not meet the actual needs of commodity circulation. PCR (polymerase chain reaction) technology is specific, sensitive, simple and rapid. Research on detecting pathogenic bacteria in food by PCR technology is ascendent. They are the four major animal food borne pathogenic bacteria including EnterohemorrhagicEscherichia coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica and Pseudomonas fluorescens. A new detection method by multiplex PCR was established in our study, combined with the universal preenrichment technique. The four species pathogenic bacteria which were contaminated in animal food can be detected at the same time by one step in 6 hours after universal preenriching with LB broth medium. The method had such advantages: well specifically, simple, rapid, economical and practical. The results were as follows:
(1) The study on the universal preenrichment method of nine species animal food borne pathogenic bacteria was effectuated. They were EnterohemorrhagicEscherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella typhi, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Shigella sonnei, and Pseudomonas fluorescens. Results showed that the optimum conditions of preenrichment were LB broth medium, cultured at 36℃for 18 h.Under these conditions, the enrichment was the best, balanced and consistent enrichment of magnitude, reaching 108 cfu /mL.)
(2) The experiment contains nine species pathogenic bacteria--EnterohemorrhagicEscherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella typhi, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Shigella sonnei,and Pseudomonas fluorescens. These bacterias were cultured under the conditions of 30℃, 33℃、36℃for 12 h, with LB,NB and UPB medium, the cell population of them can reach 106 cfu/mL. It could meet the needs of detection method, such as PCR. The culture time in 36℃should be controlled around 12 ~ 18 h, not more than 18 h.
(3) Four pairs of primer are designed in our study according to the gene: eaeA of EnterohemorrhagicEscherichia coli O157:H7, hly of Listeria monocytogenes, ail of Yersinia enterocolitica, htpX of Pseudomonas fluorescens. The result showed that four pairs of primer had good specificity.
(4) The Single PCR and multiplex PCR test system of four species pathogenic bacteria were established in our study and tested in simulation food amples , which were Enterohemorrhagic Escherichia coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica, Pseudomonas fluorescens. In the end, we have groped the optimal parameters of multiplex PCR system and thermal cycling parameters. The methods has good Specificity by a series of experiments.
引文
[1] GB/T 4789-2003.中华人民共和国国家标准.食品卫生微生物学检验[S].
[2] GB/T 4789-2008.中华人民共和国国家标准.食品卫生微生物学检验[S].
[3]韩剑众,励建荣,顾振宇,等.动物性食品卫生检验学在食品质量与安全中的地位和作用.食品与机械.2004,20(6):47-49.
[4] Jun Yatsuyanagi,Shioko Saito,Yoshimichi Miyajima,et al.Characterization of Atypical Enteropathogenic Escherichia coli Strains Harboring the astA Gene That Were Associated with a Waterborne Outbreak of Diarrhea in Japan[J].J Clin Microbiol,2003,41(5):2033–2039.
[5] Paton AW,Paton JC.Detection and characterization of shiga toxi-genic Eschenchia coli by using multiplex PCR assays for stxl stx2 enterhemorrhagic E coil hlyA,rfbO1 l1 and rfbO157[J].J Clin Microbiol,1998,36(2):598-602.
[6]柳增善.食品病原微生物学[M].北京:中国轻工业出版社,2007.
[7]郑灵巧.我科学家首次发现福氏志贺氏菌新菌型[N].健康报,2010-2-10(1).
[8]周毅,刘招丰,秦龙根.一起金黄色葡萄球菌引起食物中毒的调查[J].现代预防医学,2006,33(7):1164.
[9]朱清林,潘百斌.一起由金黄色葡萄球菌引起食物中毒事故的调查与处理[J].职业与健康, 2006,22(4):271-272.
[10]罗伟忠,谢信南,卢建聪,等.一起金黄色葡萄球菌引起食物中毒事件的调查报告职业与健康[J].2009,25(13) :1406.
[11]王滨,郑向梅,高景枝,等.一起金黄色葡萄球菌毒素引起的食物中毒[J].中国卫生检验杂志2007,17(12):2361.
[12]杨庆文,郑文康,国译丹,等.云南省2007年由金黄色葡萄球菌肠毒素A及C引起群体性食物中毒的原因调查[J].中国卫生检验杂志,2008,18(10):2135,2174.
[13]刘秀梅,陈艳,樊永祥,等.2003年中国食源性疾病暴发的监测资料分析食源性疾病的原因食品[J].卫生研究2006,35(2):201-204.
[14] Osaka K,Komatsuzaki M,Takahashi H,et al. Vibrio vulnificus septicaemia in Japan: an estimated number of infections and physicians'knowledge of the syndrome[J]. Epidemiol Infect,2004,132(5):993-996.
[15] Morimatsu Y,Akiyoshi H,Aizawa H,et al.A case of septicaemia type vibrio vulnificus infection with necrotizing fasciitis rescued by lower extremity amputation[J].Jap Assoc Infect Dis, 2003,77(3):174-177.
[16] Chung PH,Chuang SK,Tsang T,et al.Cutaneous injury and vibrio vulnificus infection [J].Emerging Infectious Diseases,2006,12(8):1302-1303.
[17]王志刚,邵平扬,吴展.创伤弧菌的微生物学特征及临床感染特点[J].中华临床感染病杂志,2008,1(5):314-317.
[18] Chang AK,Kim HY,Park JE,et al.Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolusis.J Bacteriol,2005,187(20):6909-6916.
[19] Inoue H.Vibrio vulnificus fection of the hand[J].J Orthopaedic Sci,2006,11(1):85-87.
[20] Lamps LW,Madhusudhan KT,Greenson JK,et al.The role of Yersinia enterocolitica and Yersinia pseudotuberculosis in granulomatous appendicitis:a histologic and molecular study.Am J Surg Pathol[J]. 2001,25(4):508-515.
[21] De Berardis B,Torresini G,Brucchi M,et al.Yersinia enterocolitica intestinal infection with ileum perforation:report of a clinical observation[J].Acta Biomed. 2004,75(1):77-81.
[22] Antonopoulos P,Constantinidis F,Charalampopoulos G,et al.An emergency diagnostic dilemma:a case of Yersinia enterocolitica colitis mimicking acute appendicitis in a beta-thalassemia major patient: the role of CT and literature review[J].Emerg Radiol.2007,15(2):123-126.
[23] Abdel-Haq NM,Asmar BI,Abuhammour WM,et al.Yersinia enterocolitica infection in children. [J]Pediatr Infect Dis J.2000,19(10):954-958.
[24] Koehler KM,Lasky T,Fein SB,et al.Population-based incidence of infection with selected bacterial enteric pathogens in children younger than five years of age,1996-1998[J].Pediatr Infect Dis J. 2006,25(2):129-34.
[25]于春兰,张宗一,张沈承.耶尔森菌引起儿童食物中毒调查分析[J].河北医学.1995,1(6):433-435.
[26] Tonki? M,Grgi? D,Goi?-Barisi? I, et al.A fatal case of Listeria monocytogenes sepsis and meningitis in a patient with Wegener's granulomatosis[J]. Acta Med Croatica. 2006 ,60(5):501-503.
[27] Ooi ST,Lorber B.Gastroenteritis due to Listeria monocytogenes[J].Clin Infect Dis.2005, 40(9):1327-1332.
[28]王丽芳,陈飞.冷冻鸡翼尖中单增李斯特菌的分离与鉴定[J].医学动物防制,2008,24(1): 60-62.
[29]顾振华.食品中李斯特菌进展[J].上海预防医学,2001,13(1)136-138.
[30] Okutani A., Okada Y., Yamamoto S,et al. Nationwide survey of human Listeria monocytogenes infection in Japan [J].Epidemiology and Infection ,2004, 132(4):769-772.
[31] Ericsson H,Eklow A,Danielsson-Tham ML,Loncarevic S, Mentzing LO,et al.An outbreak of listeriosis suspected to have been caused by rainbow trout[J]. J Clin Microbiol. 1997,35(11):2904-2907.
[32] Büla CJ, Bille J, Glauser MP. An epidemic of food-borne listeriosis in western Switzerland: description of 57 cases involving adults[J]. Clin Infect Dis. 1995,20(1):66-72.
[33] Dalton CB,Austin CC,Sobel J,Hayes PS,Bibb WF,Graves LM,et al.An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk[J].N Engl J Med. 1997;336(2):100-5.
[34]张芳,马国柱,潘立,等.陕西省2002~2006年食源性致病菌污染状况[J].中国公共卫生,2008,24(2):222-224.
[35]吴晓芳,程平庆,徐德顺.湖州市食品中单增李斯特菌的污染状况调查[J].中国卫生检验杂志,2007,17(10):1876-1877.
[36]母智深,白英,赵广华等.荧光假单胞杆菌胞外蛋白酶的纯化及热稳定性[J].高等学校化学学报,2008,29(4):762-766.
[37] Gilligan P H,Whittier S,Murray P R,et al.Manual of clinical microbiology[M].Washington,DC: American Society of Microbiology,1995:509-519.
[38] Sigee D C,Al-Rabaee R H.Nickel toxicity in Pseudomonas tabaci:Single cell and bulk sample analysis of bacteria cultured at high cation levels[J].Protoplasma,1986,130(2):171-185.
[39] Ghabat R,Srivastava S.Effect of zinc on morphology and ultrastructure of Pseudomonas stutzeri RS 34[J].J Gen Appl Microbiol, 1994, 40(3):265-270.
[40]冉隆贤,向妙莲,周斌.荧光假单胞杆菌的嗜铁素是控制桉树灰霉病的主要因子[J].植物病理学报,2005,35(1):6-12.
[41] Von Graevenitz A,Weinstein J.Pathogenic significance of Pseudomonas fluorescens and Pseudomonas putida[J].Yale J Biol Med,1971,44(3):265.
[42] Anderson M,Davey R.Pseudobacteraemia with Pseudomonas fluorescens[J].Med J Aust, 1994,160(4):233-234.
[43] Murray A E,Bartzokas C A,Shepherd A J,et al.Blood transfusion-associated Pseudomonas fluorescens septicaemia:is this an increasing problem[J]. J Hosp Infect,1987,9(3):243-248.
[44] Hsueh P R,Teng L J,Pan H J,et al.Outbreak of Pseudomonas fluorescens bacteremia among oncology patients.J Clin Microbiol,1998,36(10):2914-2917.
[45] Newman D J,Cragg G M.Advanced preclinical and clinical trials of natural products and related compounds from marine sources[J].Curr Med Chem,2004,11(13):1693-1713.
[46] Carvalho M F,Ferreira Jorge R,Pacheco C C,et al.Isolation and properties of a pure bacterial strain capable of fluorobenzene degradation as sole carbon and energy source[J].Environ Microbiol,2005,7(2):294-298.
[47] Jones BD, FalkowS. Salmonellosis: Host immune responses and bacteria virulence determinants[J]. Ann Rev Immunol,1996,14:533-561.
[48] Finlay BB, FalkowS. Common theme in microbial pathogenicity revisite[J]. Microbiol Mol Biol Rev,1997,61:136-169.
[49] Hacker J,Blum-OehlerG,Mühldorfer I,et al.Pathogenicity islands of virulent bacteria: structure,function and impact on microbial evolution[J].Mol Microbiol,1997,23:1089-1097.
[50] Mori H,Ito K.The Sec protein-translocation pathway[J].Trends Microbiol,2001,9:494.
[51]邱茂锋,杨瑞馥.致病性耶尔森菌毒力因YopM的研究进展[J].军事医学科学院院刊, 2004,28(6): 582-585.
[52]周宏.金黄色葡萄球菌表面蛋白研究进展[J].生物技术通讯,2004(1):73-75.
[53] Champion C S.Deivanayagam,A novel variant of the immunoglobulin fold in surface adhesins of Staphylococcus aureus:crystal structure of the Fbrinogen-bind-ing MSCRAMM, clumping factor A[J]. EMBO J,2002,21(24):6660-6672.
[54] Mamo W.Vaccination against Staphylococcus aureus mastitis : immunological response of mice vaccinated with fibronectin-binding protein (FnBP-A) to challenge with S.aureus[J].Vaccine, 1994, 12(11):988-992.
[55] Lulzim S.Immune responses to a DNA/protein vaccination strategy against Staphylococcus aureus induced mastitis in dairy cows[J].Vaccine,2004,23:114-126.
[56] Park H M.Immunogenicity of alpha-toxin,capsular polysaccharide (CPS) and recombinant fibronectin-binding protein (r-FnBP) of Staphylococcus aureus in rabbit[J].J Vet Med Sci,1999, 61(9):995-1000.
[57] Gabriel L,Development of a Staphylococcus aureus vaccine against mastitis in dairy cows.Field trial [J].Vet Immunol Immunopathol,2003,93:153-158.
[58] Garner M R,Njaa B L,Wiedmann M,et al.Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model[J]. Infect Immun,2006,74 (2): 876~886.
[59] cunliffe rn.alpha-defensins in the gastrointestinal tract.mol im-munol,2003,40(7):463-467.
[60] prouty am,brodsky ie,manos j,et al.transcriptional rogulation of salmonella enterica serouvar typhimurium genes by bile[J].fems im-munol med microbiol,2004,41(41):177-185.
[61] jepson ma,clark ma.the role of m cells in salmonella infection[J].microbes infects,2001, 3(14-15):1183-1190.
[62] H.Kitagawa,M.Hosokawa,T.Takeuchi,et al.The cellular differentiation of M cells from crypt undifferentiated epithelial cells into microvillous epithelial cells in follicle - associated epithelia of chicken cecal tonsils[J].J.Vet.Med.Sci, 2003, 65(2):171-178.
[63] acheson dw,lucciolo s.microbial-gut interactions in health and disease.mucosal immune responses[J].best pract res clin gastroenterol,2004,18(2):387-404.
[64] rescigno m,urbano m,valzasina b,et al.dendritic cells express tight junction proteins and penetrale gut epithelial monolayers to sample bacteria[J].nat immnol,2001,2(4):361-367.
[65] rescigno m,rotta g,valzasina b,et al.dendritic cells shuttle microbes across gut epithelial monolayers[J].immunobiology,2001,204(5):572-581.
[66] Sansonetti P J,Egile C.Molecular bases of epithelial cell invasion by Shigella flexneri[J].Antonie Van Leeuwenhoek,1998,74(4):191~197.
[67] Schesser K,Sp lik A K,Dakuzumuremyi JM ,et al.The yopJ locus is required for Y ersinia -mediated inhibition of N F-κB activation and cytokine expression[J].Mol Microbiol,1998,28: 1067.
[68] Yamamoto S,Okujo N,Matsuura S,et al.Siderophore-mediated utilization of iron bound to transferrin by Vibrio parahaemolyticus[J].1994, Microbiol Immunol,38: 687-693.
[69] Stevens DL . Superantigens : their role in infectious diseases[J].Immunol Invest,1997,26: 275.
[70] Thomas A,Cheasty T,Frost JA,et al.Vero cytotoxin-producing Escherichia coli,particularly serogroup O157,associated with human infections in England and ales:1992-4[J]. Epidemiol Infect,1996 Aug,117(1):1-10.
[71] Slutsker L,Ries AA,Greene KD,et al.Escherichia coli O157:H7 diarrhea in the United States:Clinical and epidemiologic features[J].Ann Intern Med,1997,126(7):505-513.
[72] National Institute of Health and Infectious Disease.Verocytotoxin-producing Escherichia coli.January 1991-November 1995,Japan[R]. Tokyo:1996.
[73] Louise CB,Obrig TG.Specific interaction of Escherichia coli O157:H7-derived Shiga-like toxin II with human renal endothelial cells[J].Infect Dis,1995,172(5): 1397–1401.
[74] Griffin P M.Escherichia coli O157:H7 and other enterohemorrhagic Escherichia coli[C].Blaser MJ,Smith PD,Ravdin JI,et al.Infections of the gastrointestinal tract[J].New York:Raven Press,1995, 739–761.
[75] Muhldorfer I,Harker J,Keusch GT,et al.Regulation of the Shiga-like toxin II operon inEscherichia coli[J]. Infect Immun,1996,664(2):495-502.
[76] Rakin A,Urbitsch P,Heesemann J,et al.Evidence for two evolutionary lineages of high pathogenic Yersinia species[J].Bacteriol,1995,177(9):2292-2298.
[77] Lindsay JA,Ruzin A,RossH F,et al.The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus[J].Mol Microbiol,1998,29(2):527-543.
[78] SN/T 1869-2007.食品中多种致病菌快速检测方法PCR法[S].
[79] SN/T 1869-2007.食品中多种致病菌快速检测方法实时PCR法[S].
[80]中国(China)食品微生物限量规定,http://down.foodmate.net/ziliao/sort/6/9319.html[S].
[81] EC 1441/2007,COMMISSION REGULATION (EC) No 1441/2007 of 5 December 2007 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs[S].
[82]黄留玉等.PCR最新技术原理、方法及应用[M].北哀化学工业出版社,2005.
[83]吴乃虎.基因工程原理(上)[M].北京:科学技术出版社,2002.
[84]徐伟,李素芳,刘军.PCR技术检测单核细胞增生李斯特氏菌研究进展[J].生物技术通报,2008,1:95-99,112.
[85]唐曙明,何林,周克元.核酸分离与纯化的原理及其方法学进展[J].国外医学(临床生物化学与检验学分册),2005,26(3):192-193.
[86]李晓虹,蒋琴娣,吴仲梁,陈家华.利用IMS/PCR方法快速检测食品中单增李斯特菌[J].检验检疫科学,2003,13(6):20-22.
[87]毛红霞,黎源倩,裴晓方,等.多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌[J].色谱,2007,l25(4): 473-477.
[88]张兰荣,唐一清,甄博珺,等.通州区6类食品单增李斯特菌污染状况及分子流行病学特征调查研究[J].中国卫生检验杂志2007,17(12):2306-2308.
[89]宫照龙,祝仁发,叶长芸,等.118株单核细胞增生李斯特菌的毒力基因检测疾病监测,2007, 22(5):299-301.
[90]杨平,杨迎伍,陈伟,等.食品中4种致病微生物的多重PCR快速检测技术研究[J].西南大学学报(自然科学版),2007,29(5):90-94.
[91]吴瑞英,黄宝明,梁均和.PCR方法检测单核细胞增多性李斯特菌的实验研究[J].中国热带医学2007,7(4):513-514.
[92]冯家望,吴小伦,王小玉,等.多重-巢式PCR检测食品中单增李斯特菌研究[J].中国国境卫生检疫杂志,2007,30(1):56-59.
[93]程洁,张晓峰,施伟良,等. SN/T 0184.2-2006食品中单核细胞增生李斯特氏菌检测方法第2部分:多重PCR方法[S].北京:中国标准出版社,2006.
[94]焦豫良,张兴群,李振勇,等.6种食品致病菌的多重PCR检测[J].临床检验杂志2005, 23(4):256-258.
[95]彭雁忠,贾宏,邹虹,等.多联实时荧光PCR定量方法检测四种肠道细菌的研究[J].中国热带医学,2005,5(5):943-947.
[96] Keiko Kimata,Tomoko Shima,Miwako Shimizu.Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR [J].Microbiol Immunol.2005,49(6),485-492.
[97] Fukushima H,Tsunomori Y,Seki R.Duplex real-time SYBR green PCR assays for detection of 17species of food or waterborne pathogens[J].J Clin Microbiol,2003;41(11):3134-3146.
[98]李善志,吴清平,张菊梅,等.RT-PCR方法检测单核细胞增生李斯特活菌研究[J].中国卫生检验杂志,2006,16(6):641-643.
[99]翁文川,杨汝德,焦红,等.免疫磁分离-荧光PCR应用在肉类单增李斯特氏菌的检测[J].中国人兽共患病学报,2006,22(6):547-550.
[100]梅玲玲,王晶,孟真,朱敏,高筱萍.TaqMan-MGB探针Real-time PCR快速检测单增李斯特菌的研究[J].中国卫生检验杂志.2007,17(2):211-213.
[101]王冰,扈庆华,石晓路,李庆阁,郑琳琳,林一曼,张顺祥,林世平,邓辉萍.食品污染产单核李斯特菌不同检测方法学评估[J].中国热带医学,2007 ,7(4):506-507,570.
[102] Vicky J,Andreja R,Johan D,et al.Kinetics of resuscitation and growth of L. monocytogenes as a tool to select appropriate enrichment conditions as a prior step to rapid detection methods[J].Food Microbiology,2009,26: 88-93.
[103] Jiang J,Larkin C,Steele M,et al. Evalution of universal preenrichment broth for the recovery of foodborne pathogens from milk and cheese[J].Journal of Dairy Science,1998, 81:2798-2803.
[104] Zhao T,Doylem P.Evaluation of universal preenrichment broth for growth of heat-in jured pathogens[J].Journal of Food Protect,2001,64(11):1751-1755.
[105] Namh M,Murinda S,Nguyen L,et al.Evaluation of universal pre-enrichment broth for isolation of Salmonellaspp.,Escherichiacoli O157:H7,and Listeria monocytogenes from dairy farm environmental samples[J]. Foodborne Pathogenic Disease,2004,1(1):37-44.
[106] Kawasaki S,Horikoshi N,Okada Y,et al.Multiplex PCR for simultaneous detection of Salmonellaspp.,Listeriamonocytogenes,and Escherichia coli O157:H7 in meat samples[J]. Journal of Food Protect,2005,68(3):551-556.
[107]王金铃.动物性食品中主要致病菌复合前增菌技术及基因芯片检测方法的建立与应用[D].长春:吉林大学,2007.
[108] Joint FAO/WHO Expert Committee on Food Additives.FAO JECFA MonograpHs 1: Combined compendium of food additive specifications [M].Rome: Food and Agriculture Organization of the United Nations,2006.
[109]慕小倩,杨超,王硕.荧光假单胞杆菌化感作用的初步研究[J].应用生态学报,2005, 16(4): 778~779.
[110] C. T. CHUNG,SUZANNE L. NIEMELA,ROGER H. MILLER.One-step preparation of competent Escherichia coli:Transformation and storage of bacterial cells in the same solution[J].Proc. Natl. Acad. Sci. USA,989,86(4):2172-2175.
[111]许文炯,丁洁,陈晓蔚等.小肠结肠炎耶尔森氏菌主要毒力基因分析[J],中国人兽共患病学报,2007,23(7):675-677.
[112]蔡亦红.PCR快速检测食品中志贺氏菌方法的建立[J] .中国人兽共患病学报2008,24(2):150-153.
