猪伪狂犬病病毒和三种主要致病菌的基因芯片检测技术研究
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摘要
随着分子生物学技术发展,许多分子生物学方法在动物疾病的诊断中得到了应用。基因芯片技术作为一项高新技术,在面积不大的载体上如玻片上可实现对多种目的基因的同时检测。它是基于PCR技术和核酸杂交的原理,事先对载体表面进行特定处理,将寡核苷酸片段或cDNA片段有序地、高密度地排列于固相载体上,形成DNA微阵列,用高精密度的激光共聚焦荧光检测系统进行杂交信号的检测。其优点为高通量、并行性、微型化、自动化、准确快速、多功能及防止污染从而有效排除外界干扰。目前国内外有许多学者都在从事基因芯片的研究工作,基因芯片技术无疑为动物疾病诊断和微生物检测提供了一项有效快速的检测技术。本试验尝试性地制备了用于检测猪伪狂犬病病毒和三种主要致病菌的基因芯片。
伪狂犬病病毒(PRV)是引起猪繁殖障碍的主要病原体之一,这种疾病给养猪业造成了巨大的经济损失,常规的病因学实验室诊断方法大多耗时长、敏感性低和需要较复杂的操作技术。本研究在分析猪伪狂犬病毒基因组结构特征的基础上,研究了PRV检测基因芯片靶基因和用于检测PRV的探针、芯片的杂交温度和时间等;将gE基因作为目的基因,并根据其基因设计引物与探针,采用不对称PCR技术扩增目的基因,将探针固定在表面经过醛基化处理过的芯片上来检测猪伪狂犬病病毒,构建了PRV病原检测基因芯片,达到了对猪伪狂犬病病毒检测的目的。
临床上常用的病原微生物诊断方法有培养技术、免疫学技术和PCR技术等。这些技术在临床诊断中发挥了巨大的作用,但仍存在一些缺点。如培养技术繁琐而费时,免疫技术要有特异的血清等;快速检测致病菌能够及时有效地预防、治疗和控制致病菌的传染。为了探索一种准确快速的检测方法,本试验尝试将基因芯片技术应用到致病菌检测领域,利用原核生物16S rRNA基因保守性兼变异性的特点,设计筛选了1对通用引物,在其保守区设计通用引物,在其变异区设计探针,实现多病原菌的检测,真正的实现高通量检测,在相同的条件下对沙门氏菌、金黄色葡萄球菌和大肠杆菌进行检测,达到了对细菌进行检测鉴定的目的,制备的基因芯片能够同时检测沙门氏菌、金黄色葡萄球菌和大肠杆菌。
With the development of molecular biology techniques, many molecular biology methods in the diagnosis of animal diseases has been applied. Microarray technology as a high-tech,in the area of small carriers such as slides can be realized simultaneously detect multiple target genes. It is based on PCR, and nucleic acid hybridization principles, prior to the carrier surface of a specific treatment, the oligonucleotide or cDNA fragment in an orderly, high-density land arranged in solid phase carrier to form a DNA microarray, with high precision degree of laser confocal fluorescence detection system for detection of hybridization signals. The advantage of high throughput, parallelism, miniaturization, automation, fast and accurate, multi-function and prevent pollution in order to effectively rule out outside interference. Many scholars at home and abroad are engaged in research on gene chip, gene chip technology has undoubtedly animal disease diagnosis and microbiological test provides a rapid and effective detection. This study tested the preparation for detection of porcine pseudorabies virus and several pathogenic gene chips.
Pseudorabies virus (PRV) is a disorder caused by porcine reproductive one of the major pathogenic bacteria,the disease to the swine industry has caused tremendous economic losses, the conventional methods of laboratory diagnosis of the etiology most time-consuming, low sensitivity and require high the operating techniques. In this study, the analysis of porcine pseudorabies virus genome structure of the basis of the PRV and the target gene microarray probes for detection of PRV, chip hybridization temperature and time; The gE gene as a target gene, and primers were designed according to the gene probe, amplified by asymmetric PCR gene, the probes immobilized on aldehyde-treated after chip up detection of pseudorabies virus, was constructed PRV pathogen microarray and reached the detection of porcine pseudorabies virus purposes.
Clinical diagnosis of pathogenic microorganisms commonly used methods of cultivation techniques, immunoassay, PCR technology, these techniques in clinical diagnosis played a huge role, but let some shortcomings. Cumbersome and time-consuming culture techniques; immune serum specific technical need, rapid detection and identification of intestinal bacteria to timely and effective prevention, treatment and control of intestinal infectious diseases. In order to explore an accurate, rapid detection method, this study attempts to gene chip technology to the field of pathogen detection, use of prokaryotic 16S rRNA gene conservation and variability of the characteristics of the design selection of the one pair of universal primers, in its conservative area design universal primers, probes in the design of its variable region, multi pathogen detection, the true high throughput detection, under the same conditions as Salmonella, Staphylococcus aureus and Escherichia coli, to detection and identification of bacteria The purpose of preparation of the gene chip can detect Salmonella, Staphylococcus aureus and Escherichia coli.
伪狂犬病病毒(PRV)是引起猪繁殖障碍的主要病原体之一,这种疾病给养猪业造成了巨大的经济损失,常规的病因学实验室诊断方法大多耗时长、敏感性低和需要较复杂的操作技术。本研究在分析猪伪狂犬病毒基因组结构特征的基础上,研究了PRV检测基因芯片靶基因和用于检测PRV的探针、芯片的杂交温度和时间等;将gE基因作为目的基因,并根据其基因设计引物与探针,采用不对称PCR技术扩增目的基因,将探针固定在表面经过醛基化处理过的芯片上来检测猪伪狂犬病病毒,构建了PRV病原检测基因芯片,达到了对猪伪狂犬病病毒检测的目的。
临床上常用的病原微生物诊断方法有培养技术、免疫学技术和PCR技术等。这些技术在临床诊断中发挥了巨大的作用,但仍存在一些缺点。如培养技术繁琐而费时,免疫技术要有特异的血清等;快速检测致病菌能够及时有效地预防、治疗和控制致病菌的传染。为了探索一种准确快速的检测方法,本试验尝试将基因芯片技术应用到致病菌检测领域,利用原核生物16S rRNA基因保守性兼变异性的特点,设计筛选了1对通用引物,在其保守区设计通用引物,在其变异区设计探针,实现多病原菌的检测,真正的实现高通量检测,在相同的条件下对沙门氏菌、金黄色葡萄球菌和大肠杆菌进行检测,达到了对细菌进行检测鉴定的目的,制备的基因芯片能够同时检测沙门氏菌、金黄色葡萄球菌和大肠杆菌。
With the development of molecular biology techniques, many molecular biology methods in the diagnosis of animal diseases has been applied. Microarray technology as a high-tech,in the area of small carriers such as slides can be realized simultaneously detect multiple target genes. It is based on PCR, and nucleic acid hybridization principles, prior to the carrier surface of a specific treatment, the oligonucleotide or cDNA fragment in an orderly, high-density land arranged in solid phase carrier to form a DNA microarray, with high precision degree of laser confocal fluorescence detection system for detection of hybridization signals. The advantage of high throughput, parallelism, miniaturization, automation, fast and accurate, multi-function and prevent pollution in order to effectively rule out outside interference. Many scholars at home and abroad are engaged in research on gene chip, gene chip technology has undoubtedly animal disease diagnosis and microbiological test provides a rapid and effective detection. This study tested the preparation for detection of porcine pseudorabies virus and several pathogenic gene chips.
Pseudorabies virus (PRV) is a disorder caused by porcine reproductive one of the major pathogenic bacteria,the disease to the swine industry has caused tremendous economic losses, the conventional methods of laboratory diagnosis of the etiology most time-consuming, low sensitivity and require high the operating techniques. In this study, the analysis of porcine pseudorabies virus genome structure of the basis of the PRV and the target gene microarray probes for detection of PRV, chip hybridization temperature and time; The gE gene as a target gene, and primers were designed according to the gene probe, amplified by asymmetric PCR gene, the probes immobilized on aldehyde-treated after chip up detection of pseudorabies virus, was constructed PRV pathogen microarray and reached the detection of porcine pseudorabies virus purposes.
Clinical diagnosis of pathogenic microorganisms commonly used methods of cultivation techniques, immunoassay, PCR technology, these techniques in clinical diagnosis played a huge role, but let some shortcomings. Cumbersome and time-consuming culture techniques; immune serum specific technical need, rapid detection and identification of intestinal bacteria to timely and effective prevention, treatment and control of intestinal infectious diseases. In order to explore an accurate, rapid detection method, this study attempts to gene chip technology to the field of pathogen detection, use of prokaryotic 16S rRNA gene conservation and variability of the characteristics of the design selection of the one pair of universal primers, in its conservative area design universal primers, probes in the design of its variable region, multi pathogen detection, the true high throughput detection, under the same conditions as Salmonella, Staphylococcus aureus and Escherichia coli, to detection and identification of bacteria The purpose of preparation of the gene chip can detect Salmonella, Staphylococcus aureus and Escherichia coli.
引文
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