鸡奇异变形杆菌致病性相关毒素检测及PCR检测方法的建立
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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.
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.
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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.
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