生物修复作用菌的竞争PCR检测方法的建立及其应用
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摘要
传统的依靠培养基进行培养、分离、生化鉴定及计数的微生物研究方法,操作繁琐、检测周期长,已远远不能满足人们对微生物快速、特异、简便、敏感且低耗的快速检测及研究的要求。近年来,微生物的快速检测和自动化研究进展迅速。快速检测及其自动化已深入到分子水平,利用核酸杂交、抗原抗体反应等对微生物进行分离、检测、鉴定和计数,使微生物的检测更为灵敏、快速、准确、方便。
竞争PCR由Gilland等[1]于1990年首先发明,其实质是用合适的内对照模板(internal control template)与待测核酸(DNA或RNA)一起竞争参与PCR反应,并通过电泳使扩增产物在凝胶上明显分开,从而进一步进行定性或定量分析。竞争PCR能消除管间及样本间的变异,定量范围较广,能够检测2倍的差异。且此方法不需要昂贵的仪器设备,实验条件容易达到。因此本论文选用竞争PCR建立了海水养殖环境生物修复作用细菌的快速定量检测方法。
竞争PCR定量方法的关键是内标,内标设计成功与否直接影响竞争PCR反应。本文通过将细菌特定DNA片段经双酶切缺失中间一小片段,将余下的2个较大片段连接,并用相同的引物在相同条件下扩增连接产物,从而获得截短的同源性片段,作为内标。制备时,将其纯化后克隆于pMD18-T载体,重组质粒经酶切和PCR鉴定,验证无误后作为竞争性模板内对照。将内标模板按一定比例稀释后分别与等体积细菌DNA进行PCR,结果表明目的DNA与竞争性DNA的扩增产物之间具有明显的线性竞争关系,结合其他细菌计数方法,建立了内标模板量与原样品中细菌数量的线性方程。本实验设计的特异性引物经PCR扩增得到了与预期PCR产物大小相当的特异性条带且敏感性较好。本竞争PCR方法的灵敏度可达到100. 5ng/μL,检测时间从平板计数需花3-5天到只需2天即能完成,大大提高了检测速度,节省了大量的时间和试剂,并且可以定量检测特定的细菌。
在建立了竞争PCR定量检测方法后,我们应用此方法检测了2006年冬季和2007年春季浙江象山港海湾网箱养殖区沉积环境中这两类菌的分布情况,五个区域分别为网箱养殖区、贝类养殖区、海带养殖区、离网箱较近的对照区、离网箱较远的对照区。检测结果表明,无论在春季样品中还是冬季样品中菌株Y都比菌株J_3多一个或两个数量级;在五个区域中网箱养殖区菌量最大,春季样品中J_3菌量为1.47×10~4个/g,Y菌量为4.59×10~4个/g;但同一区域春季与冬季相比,除离网箱较远的对照区外,同一区域J_3菌春季比冬季要多出一个数量级。Y菌除了海带养殖区可看出增加外,其余几个区域并没有明显增加。
The traditional methods, which relying on culture media to do culture, isolation, identification and counting, are complicated and consume long time, and have been far from satisfying rapid, specific, simple, sensitive and low cost requirement in detecting and studying microorganisms. In recent years, the rapid and automatic microbial detection approaches developed quickly, it has been up to molecular level by now, by using nucleic acid hybridization, PCR and antigen-antibody reactions to the isolation, detection, identification and counting microorganisms. So, Microbial detection has become more sensitive, rapid, accurate and convenient.
Competitive PCR was invented by Gilland in 1990. Overall, it uses the appropriate internal control template and the target nucleic acid (DNA or RNA) together to do the competitive PCR reaction. The PCR products can be separated by gel electrophoresis obviously, and then be further analyzed qualitatively or quantitatively. Competitive PCR can eliminate variability among samples and tubes, quantitative broader and detect the difference in 2 times. This method doesn’t need expensive equipment and it’s easy to achieve the experimental conditions. So, this thesis chooses competitive PCR to establish the rapid quantitative detection technique of functional bacteria in bioremediation in the aquatic environment.
Preparation internal control template is the key of the quantitative PCR quantitative analyses; the competitive PCR will be directly affected by the success or failure of the internal control template’s construction. In this thesis, target bacterial DNA was identified by enzymes digestion methods, two endonucleases were used to remove a small fragment in the middle, and connect the remaining two larger fragments, then the linked product was amplified in the same PCR condition using the same primers, the truncated and homologous fragments product was obtained. The product was purified and subcloned into pMD18-T vector. Restriction analysis of plasmid, PCR and DNA sequencing indicated that the competitive template had been inserted in chromosome of pMD18-T vector. Then integration plasmide DNA can be used as internal control template. After certain percentage of dilution, the internal control template was PCR amplified together with the same volume of target bacterial DNA in the same PCR reaction, the result indicated that there is distinctly competitive linear relationship between original target DNA and internal control DNA , the equation between the number of bacteria in original sample and the mass of the internal control template was set up, by doing various bacteria counting at the same time. The specificity of the primers designed for bacteria strains J_3 and Y and the sensitivity this method were also detected in this experiment. The PCR results showed the expected specific bands can be obtained. . This competitive PCR assay method is sensitive, which can detect low to 100. 5ng/μL of DNA and only need 2 days. It improves the detection rate and save a lot of time and reagents.
After the competitive quantitative PCR assay was established, it was used to test the distribution of these two types of bacteria of mud samples colleted from 5 regions of the cage culture area of Gulf in Xiangshan Port, Zhejiang Province in the winter of 2006 and spring of 2007. Five regions are the cage culture area (fish), shellfish culture area, kelp cultivation area, the nearer cage culture control plot, , farther cage culture control plot, respectively. The results show that whether in the winter samples or in the spring samples, bacteria Y are higher one or two magnitudes than J_3 bacteria. In five regions, the numbers of two kinds bacteria in the cage culture area are the highest., there were1.47×10~4cell/g J_3 bacteria and 4.59×10~4cell/g Y bacteria in spring. But in the same region, bacteria J_3 in the spring samples are one magnitude higher than those in the winter samples, except farther control plot. As to bacteria Y, significant increase of the number was observed only in the kelp cultivation area, no significant increase was observed in other regions.
竞争PCR由Gilland等[1]于1990年首先发明,其实质是用合适的内对照模板(internal control template)与待测核酸(DNA或RNA)一起竞争参与PCR反应,并通过电泳使扩增产物在凝胶上明显分开,从而进一步进行定性或定量分析。竞争PCR能消除管间及样本间的变异,定量范围较广,能够检测2倍的差异。且此方法不需要昂贵的仪器设备,实验条件容易达到。因此本论文选用竞争PCR建立了海水养殖环境生物修复作用细菌的快速定量检测方法。
竞争PCR定量方法的关键是内标,内标设计成功与否直接影响竞争PCR反应。本文通过将细菌特定DNA片段经双酶切缺失中间一小片段,将余下的2个较大片段连接,并用相同的引物在相同条件下扩增连接产物,从而获得截短的同源性片段,作为内标。制备时,将其纯化后克隆于pMD18-T载体,重组质粒经酶切和PCR鉴定,验证无误后作为竞争性模板内对照。将内标模板按一定比例稀释后分别与等体积细菌DNA进行PCR,结果表明目的DNA与竞争性DNA的扩增产物之间具有明显的线性竞争关系,结合其他细菌计数方法,建立了内标模板量与原样品中细菌数量的线性方程。本实验设计的特异性引物经PCR扩增得到了与预期PCR产物大小相当的特异性条带且敏感性较好。本竞争PCR方法的灵敏度可达到100. 5ng/μL,检测时间从平板计数需花3-5天到只需2天即能完成,大大提高了检测速度,节省了大量的时间和试剂,并且可以定量检测特定的细菌。
在建立了竞争PCR定量检测方法后,我们应用此方法检测了2006年冬季和2007年春季浙江象山港海湾网箱养殖区沉积环境中这两类菌的分布情况,五个区域分别为网箱养殖区、贝类养殖区、海带养殖区、离网箱较近的对照区、离网箱较远的对照区。检测结果表明,无论在春季样品中还是冬季样品中菌株Y都比菌株J_3多一个或两个数量级;在五个区域中网箱养殖区菌量最大,春季样品中J_3菌量为1.47×10~4个/g,Y菌量为4.59×10~4个/g;但同一区域春季与冬季相比,除离网箱较远的对照区外,同一区域J_3菌春季比冬季要多出一个数量级。Y菌除了海带养殖区可看出增加外,其余几个区域并没有明显增加。
The traditional methods, which relying on culture media to do culture, isolation, identification and counting, are complicated and consume long time, and have been far from satisfying rapid, specific, simple, sensitive and low cost requirement in detecting and studying microorganisms. In recent years, the rapid and automatic microbial detection approaches developed quickly, it has been up to molecular level by now, by using nucleic acid hybridization, PCR and antigen-antibody reactions to the isolation, detection, identification and counting microorganisms. So, Microbial detection has become more sensitive, rapid, accurate and convenient.
Competitive PCR was invented by Gilland in 1990. Overall, it uses the appropriate internal control template and the target nucleic acid (DNA or RNA) together to do the competitive PCR reaction. The PCR products can be separated by gel electrophoresis obviously, and then be further analyzed qualitatively or quantitatively. Competitive PCR can eliminate variability among samples and tubes, quantitative broader and detect the difference in 2 times. This method doesn’t need expensive equipment and it’s easy to achieve the experimental conditions. So, this thesis chooses competitive PCR to establish the rapid quantitative detection technique of functional bacteria in bioremediation in the aquatic environment.
Preparation internal control template is the key of the quantitative PCR quantitative analyses; the competitive PCR will be directly affected by the success or failure of the internal control template’s construction. In this thesis, target bacterial DNA was identified by enzymes digestion methods, two endonucleases were used to remove a small fragment in the middle, and connect the remaining two larger fragments, then the linked product was amplified in the same PCR condition using the same primers, the truncated and homologous fragments product was obtained. The product was purified and subcloned into pMD18-T vector. Restriction analysis of plasmid, PCR and DNA sequencing indicated that the competitive template had been inserted in chromosome of pMD18-T vector. Then integration plasmide DNA can be used as internal control template. After certain percentage of dilution, the internal control template was PCR amplified together with the same volume of target bacterial DNA in the same PCR reaction, the result indicated that there is distinctly competitive linear relationship between original target DNA and internal control DNA , the equation between the number of bacteria in original sample and the mass of the internal control template was set up, by doing various bacteria counting at the same time. The specificity of the primers designed for bacteria strains J_3 and Y and the sensitivity this method were also detected in this experiment. The PCR results showed the expected specific bands can be obtained. . This competitive PCR assay method is sensitive, which can detect low to 100. 5ng/μL of DNA and only need 2 days. It improves the detection rate and save a lot of time and reagents.
After the competitive quantitative PCR assay was established, it was used to test the distribution of these two types of bacteria of mud samples colleted from 5 regions of the cage culture area of Gulf in Xiangshan Port, Zhejiang Province in the winter of 2006 and spring of 2007. Five regions are the cage culture area (fish), shellfish culture area, kelp cultivation area, the nearer cage culture control plot, , farther cage culture control plot, respectively. The results show that whether in the winter samples or in the spring samples, bacteria Y are higher one or two magnitudes than J_3 bacteria. In five regions, the numbers of two kinds bacteria in the cage culture area are the highest., there were1.47×10~4cell/g J_3 bacteria and 4.59×10~4cell/g Y bacteria in spring. But in the same region, bacteria J_3 in the spring samples are one magnitude higher than those in the winter samples, except farther control plot. As to bacteria Y, significant increase of the number was observed only in the kelp cultivation area, no significant increase was observed in other regions.
引文
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