猪圆环病毒2型检测新方法研究
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摘要
猪圆环病毒2型(PCV2)可以引起很多疾病尤其是断奶仔猪多系统衰竭综合症,这种病给世界养猪业造成了巨大的经济损失。现有的PCV2的检测方法虽然对该病的诊断起到了重要作用,但难以满足现场实时快速检测的要求。因此,发展和建立PCV2早期检测新方法对于该病的检测和诊断具有重要意义。本论文结合化学发光免疫分析技术(CLIA)、乳胶凝集技术及荧光显微镜技术,采用金标单克隆抗体探针、乳胶抗体复合物探针和量子点荧光探针研究了PCV2的检测新方法,主要研究内容及结果如下：
1.合成了金纳米粒子并制备了金标单克隆抗体,对二者进行了紫外、透射电镜的表征,研究了纳米金溶解及化学发光的条件,建立了一种超灵敏、简便、快速、特异性强、重复性好的Au3+增强的CLIA检测PCV2,检测限达到2.67×102copy/mL。与PCR相比,通过检测36份临床血清样本,CLIA检测PCV2显示出了高的灵敏度和可靠性,在临床分析和控制疾病传播方面显示出了更好的前景。
2.合成了带有磺酸基的三元聚合物乳胶微球,通过静电相互作用,将PCV2单克隆抗体吸附到乳胶微球表面形成乳胶抗体复合物诊断试剂。乳胶抗体复合物诊断试剂表面抗体与病毒表面抗原发生特异性反应,促使分散的乳胶微球凝集在一起,出现肉眼可见的凝集现象,而与去离子水、磷酸缓冲液、猪圆环病毒1型、猪繁殖与呼吸障碍综合症病毒、猪细小病毒、猪传染性胃肠炎病毒不出现凝集现象。在最优化条件下,病毒浓度检测范围为3.1×105 copy/mL-8.00×107 copy/mL,检测限达到1.0×105 copy/mL。用建立的方法与PCR方法检测了34份临床血清样本,结果表明该方法可用于临床血清样品的检测,是一种简单、快速、灵敏的PCV2可视化检测方法。
3.合成了CdTe量子点,并构建了CdTe-PCV2单克隆抗体荧光探针。通过荧光光谱、紫外-可见吸收光谱、动态光散射、琼脂糖凝胶电泳、荧光显微镜等表征手段证明量子点荧光探针具有量子点的荧光特性和单克隆抗体的生物特异性。研究结果表明CdTe-PCV2单克隆抗体荧光探针在PK15细胞中可以特异性识别PCV2。
本研究将纳米探针用于PCV2检测新方法的研究,不仅提供了高灵敏、简便快速、可视化的PCV2检测方法,还为PCV2检测试剂盒的制备、PCV2致病机理的研究以及纳米探针用于其他重大动物疾病的研究提供了重要参考依据。
Porcine circovirus type 2 (PCV2) is associated with many diseases especially postweaning multisystemic wasting syndrome (PMWS), which has brought huge economic loss to the swine industry worldwide. The mainly used methods for detection of PCV2 are various kinds of biology methods. But these methods have some limits. It has great significance to develop and establish new methods for early detection of PCV2. This paper combines the chemiluminescence immunoassay, latex agglutination technique and fluorescence microscopy, using gold nanoparticle-monoclonal antibody conjugate, latex-antibody compound and quantum dot fluorescent probe for the research of detection methods and mechanism of PCV2. The main concents and results are listed as following:
1. Gold nanoparticles and gold nanoparticle-monoclonal antibody conjugate were synthetized and characterized them using UV-vis absorption spectra and TEM image. We developed a facile, rapid and ultrasensitive method for detection of PCV2 based on gold(III) enhanced chemiluminescence immunoassay (CLIA). The limit of detection was as low as 2.67×102 copy/mL. Compared with conventional polymerase chain reaction (PCR), the proposed method has good sensitivity and reliability in 36 serum samples analysis, and showed great potential in virus assay.
2. Ternary polymer latex particles with sulfonic acid group were synthesized, then we connected the monoclonal antibody of PCV2 with latex particles to obtain latex-antibody compound using electrostatic interactions. The specific reaction of monoclonal antibody and virus showed visible agglutination phenomenon in the presence of a certain concentration of virus. Latex-antibody compound didn't react with deionized water, phosphoric acid buffer, porcine circovirus type 1, porcine reproductive and respiratory syndrome virus, porcine parvovirus and porcine transmissible gastroenteritis virus. Under the optimized experimental conditions, the detection range of PCV2 was from 3.1×103 copy/mL to 8.00×107 copy/mL, and the detection limit was 1.0x105 copy/mL. Compared with PCR, the proposed method has good efficiency, sensitivity and specificity in 34 serum samples analysis. Results show that it can be used in the detection of the clinical samples and provided a great promising future for detection of PCV2 in clinical analysis.
3. CdTe quantum dots and CdTe-PCV2 monoclonal antibody fluorescent probe were synthesized and characterized them using UV-vis absorption spectra, fluorescence spectroscopy, dynamic light scattering, agarose gel electrophoresis and fluorescence microscopy. The results show that CdTe-PCV2 monoclonal antibody fluorescent probe can recognize PCV2.
In conclusion, nanoprobers were used for detection methods of PCV2. The new methods hold promising potential for sensitive, simple, rapid and visual detection of PCV2, also offers important references for the development of the detection kit and the application on the diagnostics and determination for other major animal desease of nanoprobers.
1.合成了金纳米粒子并制备了金标单克隆抗体,对二者进行了紫外、透射电镜的表征,研究了纳米金溶解及化学发光的条件,建立了一种超灵敏、简便、快速、特异性强、重复性好的Au3+增强的CLIA检测PCV2,检测限达到2.67×102copy/mL。与PCR相比,通过检测36份临床血清样本,CLIA检测PCV2显示出了高的灵敏度和可靠性,在临床分析和控制疾病传播方面显示出了更好的前景。
2.合成了带有磺酸基的三元聚合物乳胶微球,通过静电相互作用,将PCV2单克隆抗体吸附到乳胶微球表面形成乳胶抗体复合物诊断试剂。乳胶抗体复合物诊断试剂表面抗体与病毒表面抗原发生特异性反应,促使分散的乳胶微球凝集在一起,出现肉眼可见的凝集现象,而与去离子水、磷酸缓冲液、猪圆环病毒1型、猪繁殖与呼吸障碍综合症病毒、猪细小病毒、猪传染性胃肠炎病毒不出现凝集现象。在最优化条件下,病毒浓度检测范围为3.1×105 copy/mL-8.00×107 copy/mL,检测限达到1.0×105 copy/mL。用建立的方法与PCR方法检测了34份临床血清样本,结果表明该方法可用于临床血清样品的检测,是一种简单、快速、灵敏的PCV2可视化检测方法。
3.合成了CdTe量子点,并构建了CdTe-PCV2单克隆抗体荧光探针。通过荧光光谱、紫外-可见吸收光谱、动态光散射、琼脂糖凝胶电泳、荧光显微镜等表征手段证明量子点荧光探针具有量子点的荧光特性和单克隆抗体的生物特异性。研究结果表明CdTe-PCV2单克隆抗体荧光探针在PK15细胞中可以特异性识别PCV2。
本研究将纳米探针用于PCV2检测新方法的研究,不仅提供了高灵敏、简便快速、可视化的PCV2检测方法,还为PCV2检测试剂盒的制备、PCV2致病机理的研究以及纳米探针用于其他重大动物疾病的研究提供了重要参考依据。
Porcine circovirus type 2 (PCV2) is associated with many diseases especially postweaning multisystemic wasting syndrome (PMWS), which has brought huge economic loss to the swine industry worldwide. The mainly used methods for detection of PCV2 are various kinds of biology methods. But these methods have some limits. It has great significance to develop and establish new methods for early detection of PCV2. This paper combines the chemiluminescence immunoassay, latex agglutination technique and fluorescence microscopy, using gold nanoparticle-monoclonal antibody conjugate, latex-antibody compound and quantum dot fluorescent probe for the research of detection methods and mechanism of PCV2. The main concents and results are listed as following:
1. Gold nanoparticles and gold nanoparticle-monoclonal antibody conjugate were synthetized and characterized them using UV-vis absorption spectra and TEM image. We developed a facile, rapid and ultrasensitive method for detection of PCV2 based on gold(III) enhanced chemiluminescence immunoassay (CLIA). The limit of detection was as low as 2.67×102 copy/mL. Compared with conventional polymerase chain reaction (PCR), the proposed method has good sensitivity and reliability in 36 serum samples analysis, and showed great potential in virus assay.
2. Ternary polymer latex particles with sulfonic acid group were synthesized, then we connected the monoclonal antibody of PCV2 with latex particles to obtain latex-antibody compound using electrostatic interactions. The specific reaction of monoclonal antibody and virus showed visible agglutination phenomenon in the presence of a certain concentration of virus. Latex-antibody compound didn't react with deionized water, phosphoric acid buffer, porcine circovirus type 1, porcine reproductive and respiratory syndrome virus, porcine parvovirus and porcine transmissible gastroenteritis virus. Under the optimized experimental conditions, the detection range of PCV2 was from 3.1×103 copy/mL to 8.00×107 copy/mL, and the detection limit was 1.0x105 copy/mL. Compared with PCR, the proposed method has good efficiency, sensitivity and specificity in 34 serum samples analysis. Results show that it can be used in the detection of the clinical samples and provided a great promising future for detection of PCV2 in clinical analysis.
3. CdTe quantum dots and CdTe-PCV2 monoclonal antibody fluorescent probe were synthesized and characterized them using UV-vis absorption spectra, fluorescence spectroscopy, dynamic light scattering, agarose gel electrophoresis and fluorescence microscopy. The results show that CdTe-PCV2 monoclonal antibody fluorescent probe can recognize PCV2.
In conclusion, nanoprobers were used for detection methods of PCV2. The new methods hold promising potential for sensitive, simple, rapid and visual detection of PCV2, also offers important references for the development of the detection kit and the application on the diagnostics and determination for other major animal desease of nanoprobers.
引文
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