摘要
鸡奇异变形杆菌病是近年来国内新发现的一种急性热性细菌性传染病。本病的特征是呼吸困难、咳嗽、体温升高、腹泻。患病家禽主要表现为菌血症、败血症,一侧性或两侧性瘫痪或水样腹泻。各种日龄的鸡群都可感染本病,但以7周龄以下的雏鸡最易感。种鸡感染本病后可通过种蛋传给后代,从而造成雏鸡的大批死亡。此病十分容易造成爆发性流行疾病,因此对养殖业是一种潜在的威胁。
奇异变形杆菌的致病机理与其本身所含的一些毒力因子密切相关,据推测这些毒力因子可能在鸡奇异变形杆菌的病理发生和免疫方面产生重要的作用。基于上述目的本研究选取了奇异变形杆菌内毒素、热稳定性肠毒素和气管细胞毒素进行研究,初步了解奇异变形杆菌的致病机理,为研究鸡奇异变形杆菌的保护性抗原提供重要依据。
目前,有关奇异变形杆菌的检测通常采用传统的细菌分离培养、生化试验和血清学试验等多个步骤,其检测周期长、操作繁琐、灵敏度低,不适应奇异变形杆菌的快速检测。近年来发展起来的分子生物学方法克服了传统检测方法的不足。因此本研究建立了奇异变形杆菌的PCR和多重PCR检测方法,为该抗原的快速检测提供技术依据,以做到更好的控制该病的发生与蔓延。
本研究主要包括三部分:
一、鸡奇异变形杆菌相关毒素的检测及其致病性研究
本试验精提了奇异变形杆菌内毒素和粗提了热稳定肠毒素,通过动物试验研究其致病性。同时建立起鸡胚气管环模型,验证气管细胞毒素的存在及其对气管纤毛上皮的致病作用。结果表明:奇异变形杆菌内毒素可使动物呈急性败血症病变。分离的8株奇异变形杆菌有1株为ST阳性,注射ST后的乳鼠运动迟缓、食欲不振,剖检可见肠水肿,肠腔积液等。奇异变形杆菌P5、P8株分别作10-8.5、10-8.3倍稀释,可使SPF鸡胚气管环接种0.1mL后50%出现气管环纤毛脱落,气管环粘膜出现凹陷、空洞,初步验证了气管细胞毒素的存在及奇异变形杆菌对气管纤毛的损伤机理。
二、奇异变形杆菌分离株的23S rRNA序列分析
本试验通过PCR反应扩增7株鸡奇异变形杆菌和1株兔奇异变形杆菌的23S rRNA基因片段(1045bp),经克隆测序,与GenBank中收录的奇异变形杆菌、普通变形杆菌以及其它相近属的23S rRNA基因序列进行同源性比较,并由此构建系统进化发生树。本实验室保存的7株鸡奇异变形杆菌核苷酸序列与GenBank中收录的奇异变形杆菌核苷酸序列同源性为99.4%~99.8%,与兔奇异变形杆菌核苷酸序列同源性为98.8%~99.3%,与普通变形杆菌的核苷酸序列同源性为95.4%~96.2%,而与其它相近属同源性只有92.9%~93.4%。结果显示,23S rRNA基因序列分析可以作为鉴定奇异变形杆菌的一种快速、简便、准确的方法。
三、多重PCR检测三种重要食源性致病菌方法的建立及应用
本试验根据奇异变形杆菌尿素酶合成的正向调节因子R基因(ureR)、沙门氏菌侵袭性抗原保守基因(invA)和单核细胞增生性李氏杆菌编码溶血素O(LLO)的hlyA基因分别设计了三对特异性引物,对单基因PCR和单管多重PCR扩增的特异性、敏感性分析以及建立引物浓度、Tm值、模板量的L16(43)正交试验,优化单管多重PCR扩增条件,建立了快速检测奇异变形杆菌、沙门氏菌和单核细胞增生性李氏杆菌的稳定的单管多重PCR方法。结果显示,三种目的菌在105cfu/ml均可同时扩增出较清晰条带(奇异变形杆菌和单核细胞增生性李氏杆菌在104cfu/ml浓度下仍然可见到条带),比单基因PCR低一个稀释度,并且人工模拟试验和市售食品试验检测结果稳定。表明该多重PCR检测方法操作简单、快速、特异性强和灵敏度高,能够实现对奇异变形杆菌、沙门氏菌和单核细胞增生性李氏杆菌三种食源性致病菌的快速诊检和监控。
Proteus mirabilis disease is newly discovered in recent years. Features of the disease are difficulty breathing, cough, high temperature, and diarrhea. Sick chicken are mainly performance for bacteremia, sepsis, one side or both side paralysis or watery diarrhea. Chickens can be infected in any ages, and less than 7 weeks is the most susceptible time. It also can be passed to generation through the eggs, the result of which is a large number of chickens deadly. So, Proteus mirabilis is a potential threat to farming industry.
The pathogenesis of Proteus mirabilis is related to its virulence factors. Presumably, these factors may play an important role in pathology and immunity. In order to solve problems mentioned above, the study select pure endotoxin, enterotoxin, tracheal cytotoxin of Proteus mirabilis, preliminary understand the pathogenesis of Proteus mirabilis, and provide important basis for researching protective antigen of Proteus mirabilis. At present, the detection of Proteus mirabilis usually adopts the traditional bacteria separation cultivation, biochemical analysis and Serologic test, et al. Its long cycle, cockamamie operation, low sensitivity does not meet the fast detection of proteus mirabilis. In recent years, the methods of molecular biology overcome the deficiency of traditional testing method. Therefore, this study built proteus mirabilis PCR and multiple PCR detection method, provide technical support for the rapid detection, and better control the occurrence and spread of this disease.
The research divides three parts:
Part One: Extraction and detection of biological characteristics of Proteus mirabilis related toxins
Pure endotoxin and heat-stable enterotoxin of Proteus mirabilis were prepared to study pathogenicity by animal test At the same time, chicken embryo tracheal ring model was established to verify the existence of tracheal cytotoxin and its pathogenic effects on tracheal cilia epithelium. The results showed that: endotoxin of Proteus mirabilis can effectively lead to acute septicemia lesions of animals. One of eight strains Proteus mirabilis produces ST, the little mousse injected ST are sluggishness, in appetence, cutting inspection can see bowel oedema, fluctuate effusion, etc. When the strains (Q3, Q5) Proteus mirabilis were diluted 10-8.5 and 10-8.3 times , abscission and injury of 50% of chicken embryo tracheal rings cilia could be caused by inoculating 0.1mL Proteus mirabilis. This result preliminarily verified the existence of tracheal cytotoxin and revealed pathogenic mechanism of Proteus mirabilis on tracheal cilia. The biological characteristics of these toxins played an important role for studying Proteus mirabilis vaccines.
Part Two: Sequence analysis of 23S rRNA genes of Proteus mirabilis
Establish the sequence analysis of 23S rRNA of Proteus mirabilis for the identification of Proteus mirabilis strains. The fragments (1045bp) of 23S rRNA genes of seven chicken Proteus mirabilis strains and one rabbit Proteus mirabilis strains were amplified by PCR and were sequenced. The sequences obtained in this paper were compared with the Proteus mirabilis 23S rRNA sequence reported in GenBank and other similar generas by the software of DNAStar analyses. Construct the Phylogenetic tree of Proteus mirabilis strains. Results showed the homologies between seven chicken Proteus mirabilis strains and the reported one strain in GenBank were 99.4%-99.8%, and were 98.8%-99.3% identical to the rabbit Proteus mirabilis strains, were 95.4%-96.2% identical to Proteus vulgaris, were 92.9%-93.4% identical to other similar generas. The result shown that 23S rRNA sequence analyses may be a reliable and rapid way for identification of Proteus mirabilis.
Part Three:Establishment and application of multiple PCR for diagnosing Proteus mirabilis,Salmonella and Listeria monocytogenes
Based on the gene sequences of positive adjustment factor R gene of Urease (ureR) in Proteus mirabilis, conservative invasive antigen gene (invA) in Salmonella and the hly gene in Listeria monoytogenes, a three pairs of primer were designed. The specificity and sensitivity of PCR were analyzed for single gene. Multiple PCR method has been developed after analysis and optimization reaction condition by orthogonal experimental design L16 (43). The result shows, under the optimized conditions, the anticipated PCR of Proteus mirabilis, Salmonella and Listeria monocytogenes were specificity highly, and the minimum detection limit was 105 cfu/ml, which are low a dilute degrees in the sensitivity of single PCR. The results were stable in simulated examination and in marketing examination. A rapid, specific and sensitive multiplex PCR system has been established and it is valuable for diagnosis and monitoring for Proteus mirabilis, Salmonella and Listeria monocytogenes.
引文
陈炳卿,刘志诚,王茂起.现代食品卫生学.人民卫生出版社, 2001, 771.
陈金顶,索青利,廖明,等.沙门氏菌的invA基因序列分析与分子检测.中国人兽共患病杂志, 2004, 20(10): 868-871.
陈文炳,邵碧英,李寿崧,等.应用多重PCR同时检测多种转基因成分.检验检疫科学, 2002, 12(3): 11-13.
崔晓燕,陈峰,谭湘凌,等.食源性致病菌多重聚合酶链反应快速检测.现代预防医学, 2010, 37(3): 519-520.
董关萍. 23S rRNA基因在临床诊断细菌性感染中的应用.国外医学流行病学传染病学分册, 2002, 4(29): 103-105.
范宏英,吴清平,吴若著.饮用水中5种致病菌多重PCR技术检测研究.微生物学通报, 2005, 32(3): 102-107.
郜红梅,高洁,张弛.一起奇异变形杆菌引起的食物中毒调查分析.安徽预防杂志, 2008, 14(3): 219-220.
郭新竹,宁正祥. PCR技术在食品检验中的应用.广州食品工业科技, 2001, 17: 60-62.
黄留玉,王恒梁,史兆兴,等. PCR最新技术原理、方法及应用.北学工业出版社, 2005.
洪帮兴,江丽芳,胡玉山,等. 23S rRNA基因序列分析及其在细菌鉴别诊断中的应用.中华微生物和免疫学杂志, 2004, 24(3): 241-244.
江益民,秦翠丽,龙塔,等.鸡奇异变形杆菌病的研究.豫西农专学报, 1990, (1): 7-9.
李鹏,马艳娇,赵云,等. 16S rRNA、23S rRNA及16S~23S rRNA基因在细菌分离与鉴定中的应用.现代畜牧兽医, 2008(7): 49-52.
李文建,于长青,李爱华,等.慢性腹泻与肠道需氧优势菌群的关系.中国人兽共患病杂志, 1998, 14(5): 56.
李业鹏,钟凯.食品中沙门氏菌PCR检测方法的建立.中国食品卫生杂志, 2006, 18(1): 17-22.
林海.中山市变形杆菌食物中毒现况分析.中国卫生监督杂志, 2005, 12(4): 270-271.
林红乐,文岚,张如胜. PCR快速检测奇异变形杆菌的研究.实用预防医学, 2009, 16(2): 560-562.
林东,徐晓刚,朱德妹,等. 23S rRNA基因在常见病原菌鉴定中的应用.中华检验医学杂志, 2005, 28(1): 79-81.
刘红芹,朱瑞良,胡晓娜,等.禽波氏杆菌分离株的16S rRNA基因序列分析.中国兽医学报, 2008, 28(1): 32-34.
刘俊康,王源,黄春基,等.细菌波动潜生体增殖方式的观察研究.中华流行病学杂志, 1996, 17(3-c): 134.
刘明辉,陈建琳,等.变形杆菌食物中毒生物模式初探.中国卫生检验杂志, 2000, 12, 10(6): 714-715.
刘渠,甘莉萍.变形杆菌引起食物中毒的初步研究.中国卫生检验杂志, 1998, 8(4): 208-210.
龙塔,程相朝,江益民,等.鸡奇异变形杆菌病病理学研究.中国畜禽传染病, 1997, 4: 15-18.
陆承平.兽医微生物学[M] (第三版).北京,中国农业出版社, 2002: 237-238.
罗新茂,冯新萍.一起由奇异变形杆菌引起食物中毒的调查.现代预防医学, 2006, 33(7): 1157-1158.
彭丽萍,冯维占.从进口冻鸡副产品中检出奇异变形杆菌.动植物检疫, 1999(2): 26.
钱玉春,类延花,胡中. 4种食源性致病菌的多重PCR检测方法的建立.中国卫生检验杂志, 2009, 19(9): 2042 -2045.
孙健. 2003~2007年细菌性食物中毒病原菌检测结果分析.现代预防医学, 2008, 35(19): 3702-3703.
谌南辉,邓舜洲,郭松林,等.珍禽源奇异变形杆菌分离株微生物学特征研究.中国预防兽医学报, 1999, 21(4): 18-22.
孙秋艳,刘红芹,朱瑞良,等.鸡奇异变形杆菌的分离、鉴定及流行病学调查.家畜生态学报, 2006, 27(6): 115-119.
沈正达.细菌毒力岛的研究.中国兽医学, 2003, 23(4): 412- 414.
尚世强,傅君芬,洪文澜.葡萄球菌属16S-23S rRNA基因区间的图谱研究.中华检验医学杂志, 2001, 24: 308.
王春平,韦强,鲍国连,等.鸭疫里默氏菌病和大肠杆菌病多重PCR诊断方法的建立.中国兽医学报, 2010, 30(3): 352-355.
王福.一起因食用烧鸡引起的奇异变形杆菌食物中毒的调查报告.口岸卫生控制, 2003, 8(l): 55.
王恒安.雏鸡奇异变形杆菌病的诊断及防治.黑龙江畜牧兽医, 2001(8): 24-25.
徐文杰,侯兰.奇异变形杆菌致病因素检测.中国卫生检验杂志. 2001, 11(2): 206-206.
杨瑞军,金莞尔,张建民,等.生肉食品中致病菌检测结果分析.中国卫生检验杂志, 2009, 19(9): 2122-2125.
曾海燕,张晓峰.三重PCR鉴定消毒牛奶及其加工环境中的单核细胞增生李斯特菌.青海医学院学报, 2006, 27(2): 75-50.
张辉,崔焕忠,何昭阳.应用PCR检测单核细胞增生性李斯特氏菌.中国兽医杂志, 2007,9(43): 76-77.
张玉霞,黄鸣.食品检验中多重PCR技术的应用.中国卫生检验杂志, 2008, 1 8(5): 958-960.
周霞,刘玉娥.嗜碱耐盐奇异变形杆菌的分离与鉴定及致病性研究.职业与健康, 2002, 18(11): 32-34.
周有森. 35例细菌性食物中毒分析.临床医学工程, 2009, 16(9): 95-96.
朱瑞良,张绍学,牛钟相. AA肉种鸡奇异变形杆菌病流行病学调查.中国畜禽传染病, 1994, 77 (4): 46-47.
朱庆义.现代分子生物学技术在病原微生物快速诊断中的应用.中华检验医学杂志, 2003, 26: 737-740.
Abd E I, Asiz T. Screening of some food Poisoning bacteria in sausage and hamburger meat[J]. Egypt Public Health Assoc, 1996(71): 47-61.
Andrea Germini, Annalisa Masola, Paola Carnevali, et al. Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR Food Control, 2009, 20(8): 733-738.
Anrhony R M, Brown T J, French G L. Rapid diagnosis of bacteria by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array [J]. J Clin Microbio, 2000, 38: 781-788.
Aznar R, Alarcon B. On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers. Systematic and Applied Microbiology, 2002, 25(1): 109-119. Banning M. Bacteria and the gastrointestinal tract: beneficial and harmful effects. Br J Nurs, 2006 (3): 144-149.
Barsotti O, Decoret D, Renaud FN. Identification of streptococcusmitis group species by RFLP of the PCR amplified 16S-23S rDNA intergenic spacer. Res Microbiol, 2002, 153: 687-691.
Barry T, Colleran G, Glennon M, et al. The 16s/ 23s ribosomal spacer region as a target for DNA probes to identify eubacteria. PCR Methods Appl, 1991, 1:51-56.
Belas R, Erskine D, Flaherty D. Proteus mirabilis mutant’s defeetive in swarmer cell differentiation and multicellular behavior. J Bacteriol, 1991,173 (19):62-79. Belas R. The swanning Phenomenon of Proteus mirabilis. ASM News, 1992, 58 (l): 15.
Chamberlain J, Gibbs R A. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleid Acids Research, 1988, 16: 11141- 11156.
Chotár M, VidováB, Godány A, et al. Development of specific and rapid detection of bacterialpathogens in dairy products by PCR. Folia Mi-crobiol, 2006, 51(6): 639-646
CS Aman, A Pastor, G Cighetti, et al. Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC-MS/MS. Anal Bioanal Chem, 2006, 386: 1869-1879.
Daniel B, Liarte, Silvane M.F., Murta, Mario Steindel and Alvaro J. Romanha. Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II. Experimental Parasitology, 2009, 123(4): 283-291.
Drechsel H, Thieken A, Reissbrodt R, Jung G and Winkelmann G Alpha-keto acids are noel siderophores in the genera Proteus Providencia. And Morganella and areproduced by amino acid dcaminaese. Journal of Bacteriology, 1993, 175(9): 2727-2733.
Elfath E M, Ahmed A M, Cooper R J, et al. Multiplex PCR: Optimization and application in diagnostic virology. Clinical Microbiology Reviews, 2000(10): 559-570.
EMIKO Rimbara, NORIHISA Noguchi, HIDENOBU Kijima, et al. Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan. International Journal of Antimicrobial Agents, 2007, 30(3): 250-254.
Enrica Omiccioli, Giulia Amagliani, Giorgio Brandi and Mauro Magnani. A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk. Food Microbiology, 2009, 26(6): 615-622.
Erol I, Goncuoglu M, Ayaz ND, Bilir Ormanci FS, Hildebrandt. Molecular typing of Clostridium perfringensi solated from turkey meat by multiplex. Lett Appl Microbiol. 2008, l47 (1):31-34
GeorgeM, Garrity. Bergey’s Manual of Systematic Bacteriology and Edition. Bergey’s Manual Trust, 2004.
Gurtler V, Stanisich V A. New approaches to typing and identification of bacteria using 16S-23S rDNA spacer region. Microbiol, 1996(142): 3-16.
Hansen B M, Hendriksen N B. Detection of enterotoxic bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Appl Environ Microbiol, 2001, 67(1): 185-189.
Hirohito Ogawa, Osamu Taira, Takuya Hirai, Hiromi Takeuchi, Aki Nagao, Yoshiki Ishikawa, Kotaro Tuchiya, Tetsuo Nunoya and Susumu Ueda. Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections. Journal of Virological Methods, 2009, 106(1-2):210-214.
Jens P, Fannebecker, and Jurgen Frohlich. Use of a species-specific multiplex PCR for the identification of pediococci. International Journal of Food Microbiology, 2008, 128 (2): 288-296.
Kawasaki S,Horikoshi N,Okada Y. Multiplex PCR of simultaneous detection of Salmonella spp.,Listeria monocytogenes, and Escherichia coli 0157:H7 in meat samples. Journal of Food Protect, 2005, 68(3): 551-556.
Kim C H, Khan M, Morin D E, et al. Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine. J Dairy Sci, 2001, 84(1): 74-83.
Ki m J, Demeke T, Clear RM, et al. Simultaneous detection by PCR of Escherichia coli, L isteria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. International Journal of Food Microbiology, 2006, 111(1): 21-25.
[Kong R Y, Lee SKY, Law T W, et al. Rapid detected of six types of bacterial pathogens in marine waters by multiplex PCR. Water Res, 2002, 36(11):2802-2812.
Li X, Boudjellab N, Zhao X. Combined PCR and slot assay for detection of Salmonella and Listeriamonocytogenes. International Journal of food Microbiology, 2000, 56(1): 167-177.
LimY H,Hirose K, Izumiya H. Multiplex Polymerase chain reaction assay of for selective detection of salmonella enterica serovar typhimurium. Japanese Journal Infect Disease, 2003, 56: 151-155.
Li Y, Zhang S, Mustapha A. Application of a multiplex PCR for the simultaneous detection of Escherichia coli 0157:H7,Salmonella and Shigella in raw and ready-to-eat meat products. Meat Science, 2005, 71: 402-406.
Ludwig W, Rossello R, Aznar R, et al. Comparative sequence analysis of 23S rRNA from proteobacteria. Syst Appl Microbiol, 1995(18): 164-188.
Mahua D, Anderson, and Joseph M. Development of a multiplexed PCR detection method for Barley and Cereal yellow dwarf viruses, Wheat spindle streak virus, Wheat streak mosaic virus and Soil-borne wheat mosaic virus. Journal of Virological Methods, 2008, 142(1-2): 17-24.
Mansy MS, Fadl AA, Ashour MS, et al. Amplification of Proteus mirabilis chromosomal DNA using the polymerase chain reaction. Mol Cell Probes, 1999, 13(2): 133-140.
Mekonnen K, Divind E, Ruth-Anne S, et al. Amultiplex polymerase chain reaction assay for genus group and species-specific detection of mycobacteria. Diag Microbiol Infect Dis, 2004, 49:99-104.
Malorny B, Tassios P T, Radstrom P, et al. Standardization of diagnostic PCR for the detection of foodborne pathogens. International Journal of Food Microbiology, 2003, (5):39-48.
Newcombe J, Cartwright K, Palmer W H, et al. PCR of peripheral blood for diagnosis meningococcal disease. J Clin Microbiol, 1996(134): 1637- 1640.
NILTON Lincopan, LARA M. de Almeida, MARIA R. Elmor de Araújo, et al. Linezolid resistance in Staphylococcus epidermidis associated with a G2603T mutation in the 23S rRNA gene. International Journal of Antimicrobial Agents, 2009, 34 (3): 281-282.
Nurhan Ertas, Zafer Gonulalan, Yeliz Yildirim, et al. Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique. International Journal of Food Microbiology, 2010, 142(1-2): 74-77.
O’Hara C.M., Brenner F.W.and Miller J.M. Classification, identification and clinical significance of Proteus.Providencia.and Morganella. Clinical Mircrobiology Reviews, 2000, 13(4): 534-546.
Ooka T, Terajima J, Kusumoto M, Iguchi A, Kurokawa K, Ogura Y, Asadulghani M, Nakayama K, Murase K, Ohnishi M, Iyoda S, Watanabe H, Hayashi T. Development of a multiplex PCR-based rapid typing method for enterohemorrhagic Escherichia coli O157 strains.J Clin Microbiol, 2009, 47(9): 2888-2894.
Park Y S, Lee S R, Kim Y G. Detection of Escherichia coliO157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in Kimchi by multiplex polymerase chain reaction (mPCR). Journal of Microbiolog February, 2006, 44(1): 92-97.
Patel R, Piper KE, Rouse MS, et al. Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates. J Clin Microbiol, 1998, 36: 3399-3407.
Peter M. Vallone, Carolyn R, Hill, and John M. Butler. Demonstration of rapid multiplex PCR amplification involving 16 genetic loci. Forensic Science International: Genetics, 2008, 3(1):42-45.
Rajkhowa S, Das R, Bora S, Rajkhowa C, Rahman H, Bujarbaruah KM. Detection of ShigaToxin-Producing Escherichia coli and Enter pathogenic Escherichia coli in Faecal Samples of Healthy Mithun (Bos frontalis) by Multiplex Polymerase Chain Reaction. Zoonoses Public Health. 2009, 23(5): 34-40.
Ramesh A, Padmapriya B P, Chandrasekhar A, et al. Application of a convent DNA extraction method and multiplex PCR for the direct of Staphylococcus aurous and Yesinia enterocolitica in milk samples. Molecular and Cellular Probes, 2002, 16: 307-314.
Roller C, Ludwig W, Schleifer K H. Gram- positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA. J Clin Mi-crobiol, 1992(138): 1167- 1175.
Senior B W. Proteus morgani is less frequently associated with urinary tract infeetions than Proteus mirabilis-an explanation. J Med Microbiol, 1983, 16(4): 31.
Sobiraj A, Kron A. Investigations on the distribution and in vitro resistance of udder Pathogenic bacteria in the milk of cows with subclinical mastitis. Tierarztl Prax, 1997, 25(2): 108-115.
Sosa V,Schlapp G, Zunino P. Proteus mirabilis isolates of different origins donot show Correlation with virulence attributes and can colonize the urin ary tract of Mice. Microbiology, 2006(152):2149-2157.
Soumet C,Emrel G,Rose V. Identification by a multiplex PCR- based assay of Salmonella Typhimurium and Salmonella Enteritdis strains from environmental swabs of Poultry houses. Letters in Applied Microbiology, 1999, 29:1-6.
Tandir S. Health accuracy of Travnik cheese and quality standards of sanitary accuracy. Med Arh, 2005, 59(5): 326-327.
Tsiodras S, Gold HS, Coakley EP, et al. Diversity of domain V of 23S rRNA gene sequence in different Enterococcus species. J Clin Microbiol, 2000, 38: 3991-3993.
Vantarakis A, Komninou G, Venieri D, et al. Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels. Letters in Applied Microbiology, 2000, 31: 105-109.
Wang X, Jothikumar N, Griffiths MW. Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR. Journal of Food Protection, 2004, 67(1): 189-192.
Wang Y, Zhang Z S. Comparative sequence analyses reveal frequent occurrence of short segments containing an abnormally high number of nonrandom base variations in bacterial rRNAgenes. Microbiol, 2000(146): 2845-2854.
Wilson C, Thakore A, Isenberg D, et al. Correlation between anti–pr oteus antibodies and isolation rates of P.mirabilis in rheumatoid arthri tis. Rheumatol Int,1997,16(5):187-189.
Wilson KH, Blitchington R B, Greene R C. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol,1990, 28: 1942-1946.
Woese C R. The universal ancestor. Proc Natl Acad Sci, 1998(95): 6854-6859.
Yamamoto T, Nakazawa M J. Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene inenterotoxigenic E. coli strains isolated from piglets and calves withdiarrhea. ClinMicr obiol, 1997, 35 (1): 223-227.
Yenny Suanthie, Maribeth A, Cousin and Charles P. Woloshuk. Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium and Fusarium. Journal of Stored Products Research, 2009, 45(2): 139-145.