猪圆环病毒快速检测技术的研究
摘要
猪圆环病毒2型(PCV2)是近年来发现严重影响养猪业的一种最小的DNA病毒,是断奶仔猪多系统衰竭综合征的主要病原。目前报道检测PCV2抗原的方法有病毒分离、聚合酶链反应技术、免疫组化实验(IHC)、间接免疫荧光(IFA)、原位杂交技术等。然而,这些检测方法均未系统化、标准化,难以推广应用。同时没有快速简便的病原检测技术。本研究就PCV2分子生物学检测方法与免疫荧光方法进行了比较,研究了猪圆环病毒2的胶体金试纸条技术,为寻找适合现场使用的PCV2检测方法奠定了基础。
用新生牛血清和标准PCV2阴性猪血清分别稀释1766PCV2细胞适应毒21代,使其终浓度分别为1.0×107 TCID50/ml, 1.0×106 TCID50/ml, 1.0×105 TCID50/ml, 1.0×104 TCID50/ml, 1.0×103 TCID50/ml, 1.0×102 TCID50/ml, 1.0×101 TCID50/ml, 1.0×100 TCID50/ml, 1.0×10-1 TCID50/ml。然后分别用DNA试剂盒抽提病毒DNA、进行普通PCR和实时荧光定量PCR检测。普通PCR检测显示,新生牛血清和猪标准阴性血清稀释的PCV2样本可检测到PCV2抗原的灵敏度均是1.0×103TCID50/ml(约为5.04×104个拷贝/ml)。实时荧光定量PCR检测显示,新生牛血清和猪标准阴性血清稀释的PCV2样本中可检测到的PCV2抗原的灵敏度均是1.0×101TCID50/ml(约5.23×102个拷贝/ml)。细胞病毒分离检测显示,当新生牛血清稀释的样本在PK15细胞培养时,第3代可检测到的PCV2抗原的检测灵敏度是1.0×101TCID50/ml；而当猪血清稀释的样本在PK15细胞培养时,第3代可检测到的PCV2抗原得灵敏度是1.0×103TCID50/ml。这些结果说明,细胞培养病毒分离法对猪血清中PCV2的分离能力低于对牛血清中PCV2病毒分离能力的100倍,实时荧光定量PCR是实验室检测猪血清样本中PCV2的抗原首选方法。
用柠檬酸三钠还原法制备的金溶胶对纯化的单克隆抗体PCV2-ORF2-5E11、PCV2-ORF2-8A12、PCV2-ORF2-6H9和PCV1-ORF2-3F11进行标记,结果显示,胶体金标记单克隆抗体IgG的最佳工作浓度是20ng/ml胶体金。以亲和层析纯化的猪抗PCV2 IgG为检测线、羊抗鼠IgG为质控线、以胶体金标金的单克隆抗体为抗体与待检抗原反应。用Millpore 135硝酸纤维素膜和Ahlstrom 8964玻璃纤维素膜在Bio-dot三维喷点仪上制备试纸条,结果显示,金标PCV2-ORF2-6H9和PCV2-ORF2-8A12的两种试纸条可快速、特异性的检测基因组大小分别1766bp和1767bp的PCV2。金标PCV2-ORF2-6H9的试纸条对基因组大小为1766bp的PCV2病毒的检测灵敏度是1.0×107TCID50/ml,而对基因组大小为1767bp的PCV2病毒的检测灵敏度是1.0×106 TCID50/ml;金标PCV2-ORF2-8A12的试纸条对基因组大小为1767bp的PCV2的检测灵敏度是1.0×108TCID50/ml。试纸条在4℃或室温的有效保存期达12个月以上。
Porcine circovirus type 2 (PCV2) is the main pathogen associated with post-weaning multisystemic wasting syndrome. Several detection methods for porcine circovirus have been reported, i.g. viral isolation, indirect immunofluorescence assay (IFA), Immunohistochemistry (IHC), in situ hybridization (ISH), PCR and so on. But these methods have not been systematized and standarded, so it is hard to apply in veterinary practice. There also have no quick and convenient antigen detection method for PCV. The objectives of this study are to standardize a molecular Diagnostic method and to develop a new immunochromatographic strip for PCV2 detection.
In this research, using respectively new-born calf serum and norm swine PCV2-negative serum, PCV2 particles were diluted to various final concentration at 1.0×107 TCID50/ml,1.0×106 TCID50/ml, 1.0×105 TCID50/ml, 1.0×104 TCID50/ml,1.0×103TCID50/ml,102TCID50/ml, 10TCID50/ml, 1TCID50/ml and 1.0×10-1 TCID50/ml. Genomic DNA of PCV2 were extracted by the DNA extract kit to perform routine PCR and real-time quantitation PCR (qPCR) detection. The result of routine PCR shows that the least detective limit is 1.0×103TCID50/ml (approximately 5.04×104 copies/ml) for PCV2 in new-born calf serum and norm swine PCV2-negative serum. The result of real-time PCR shows that the the least detective limit is 1.0×101TCID50/ml (approximately 5.23×102 copies/ml) for PCV2 in new-born calf serum and norm swine PCV2-negative serum. IFA shows that the least detective limit is 1.0×101TCID50/ml for PCV2 in new-born calf serum, 1.0×103 TCID50/ml for PCV2 in swine serum, when was generated three times in PK15 cell. These data confirm that PCV2 isolation in new-born calf serum is more convenient than in swine serum and real time quantitation PCR is the preferential method to detect the PCV2 antigen in swine serum.
The mAbs 8A12,5E11 and 6H9 specific for PCV2-ORF2, and 3F11 specific for PCV1-ORF2 and swine anti-PCV2 serum were purified by the protein G. The colloidal gold suspension was generated by trisodium citrate decompose. The result shows that the optimal working concentration is 20ng/ml colloidal gold when the mAb was labelled. The test line is anti-PCV2 IgG and the control line is goat-anti-mouse IgG. Then using the nitrocellulose filter and Ahlstrom 8964 cellulose membrane, the lateral flow strip was prepared in the XYZ 3000 platform with BioJet Quanti 3000 and AirJet Quanti 3000 dispensers (BioDot Inc., Irvine, CA). At last, the lateral flow strips loaded the 6H9 and the 8A12 can detect the PCV2 particles. And the limit concentration of the 6H9-marked lateral flow strip is 107TCID50/ml for 1766PCV2,106TCID50/ml for 1767PCV2. The limit concentration of the 8A12-marked lateral flow strips is 108TCID50/ml for 1767PCV2. The expiration of the lateral flow strips is 12 monthes at 4℃or at room-temperature.
用新生牛血清和标准PCV2阴性猪血清分别稀释1766PCV2细胞适应毒21代,使其终浓度分别为1.0×107 TCID50/ml, 1.0×106 TCID50/ml, 1.0×105 TCID50/ml, 1.0×104 TCID50/ml, 1.0×103 TCID50/ml, 1.0×102 TCID50/ml, 1.0×101 TCID50/ml, 1.0×100 TCID50/ml, 1.0×10-1 TCID50/ml。然后分别用DNA试剂盒抽提病毒DNA、进行普通PCR和实时荧光定量PCR检测。普通PCR检测显示,新生牛血清和猪标准阴性血清稀释的PCV2样本可检测到PCV2抗原的灵敏度均是1.0×103TCID50/ml(约为5.04×104个拷贝/ml)。实时荧光定量PCR检测显示,新生牛血清和猪标准阴性血清稀释的PCV2样本中可检测到的PCV2抗原的灵敏度均是1.0×101TCID50/ml(约5.23×102个拷贝/ml)。细胞病毒分离检测显示,当新生牛血清稀释的样本在PK15细胞培养时,第3代可检测到的PCV2抗原的检测灵敏度是1.0×101TCID50/ml；而当猪血清稀释的样本在PK15细胞培养时,第3代可检测到的PCV2抗原得灵敏度是1.0×103TCID50/ml。这些结果说明,细胞培养病毒分离法对猪血清中PCV2的分离能力低于对牛血清中PCV2病毒分离能力的100倍,实时荧光定量PCR是实验室检测猪血清样本中PCV2的抗原首选方法。
用柠檬酸三钠还原法制备的金溶胶对纯化的单克隆抗体PCV2-ORF2-5E11、PCV2-ORF2-8A12、PCV2-ORF2-6H9和PCV1-ORF2-3F11进行标记,结果显示,胶体金标记单克隆抗体IgG的最佳工作浓度是20ng/ml胶体金。以亲和层析纯化的猪抗PCV2 IgG为检测线、羊抗鼠IgG为质控线、以胶体金标金的单克隆抗体为抗体与待检抗原反应。用Millpore 135硝酸纤维素膜和Ahlstrom 8964玻璃纤维素膜在Bio-dot三维喷点仪上制备试纸条,结果显示,金标PCV2-ORF2-6H9和PCV2-ORF2-8A12的两种试纸条可快速、特异性的检测基因组大小分别1766bp和1767bp的PCV2。金标PCV2-ORF2-6H9的试纸条对基因组大小为1766bp的PCV2病毒的检测灵敏度是1.0×107TCID50/ml,而对基因组大小为1767bp的PCV2病毒的检测灵敏度是1.0×106 TCID50/ml;金标PCV2-ORF2-8A12的试纸条对基因组大小为1767bp的PCV2的检测灵敏度是1.0×108TCID50/ml。试纸条在4℃或室温的有效保存期达12个月以上。
Porcine circovirus type 2 (PCV2) is the main pathogen associated with post-weaning multisystemic wasting syndrome. Several detection methods for porcine circovirus have been reported, i.g. viral isolation, indirect immunofluorescence assay (IFA), Immunohistochemistry (IHC), in situ hybridization (ISH), PCR and so on. But these methods have not been systematized and standarded, so it is hard to apply in veterinary practice. There also have no quick and convenient antigen detection method for PCV. The objectives of this study are to standardize a molecular Diagnostic method and to develop a new immunochromatographic strip for PCV2 detection.
In this research, using respectively new-born calf serum and norm swine PCV2-negative serum, PCV2 particles were diluted to various final concentration at 1.0×107 TCID50/ml,1.0×106 TCID50/ml, 1.0×105 TCID50/ml, 1.0×104 TCID50/ml,1.0×103TCID50/ml,102TCID50/ml, 10TCID50/ml, 1TCID50/ml and 1.0×10-1 TCID50/ml. Genomic DNA of PCV2 were extracted by the DNA extract kit to perform routine PCR and real-time quantitation PCR (qPCR) detection. The result of routine PCR shows that the least detective limit is 1.0×103TCID50/ml (approximately 5.04×104 copies/ml) for PCV2 in new-born calf serum and norm swine PCV2-negative serum. The result of real-time PCR shows that the the least detective limit is 1.0×101TCID50/ml (approximately 5.23×102 copies/ml) for PCV2 in new-born calf serum and norm swine PCV2-negative serum. IFA shows that the least detective limit is 1.0×101TCID50/ml for PCV2 in new-born calf serum, 1.0×103 TCID50/ml for PCV2 in swine serum, when was generated three times in PK15 cell. These data confirm that PCV2 isolation in new-born calf serum is more convenient than in swine serum and real time quantitation PCR is the preferential method to detect the PCV2 antigen in swine serum.
The mAbs 8A12,5E11 and 6H9 specific for PCV2-ORF2, and 3F11 specific for PCV1-ORF2 and swine anti-PCV2 serum were purified by the protein G. The colloidal gold suspension was generated by trisodium citrate decompose. The result shows that the optimal working concentration is 20ng/ml colloidal gold when the mAb was labelled. The test line is anti-PCV2 IgG and the control line is goat-anti-mouse IgG. Then using the nitrocellulose filter and Ahlstrom 8964 cellulose membrane, the lateral flow strip was prepared in the XYZ 3000 platform with BioJet Quanti 3000 and AirJet Quanti 3000 dispensers (BioDot Inc., Irvine, CA). At last, the lateral flow strips loaded the 6H9 and the 8A12 can detect the PCV2 particles. And the limit concentration of the 6H9-marked lateral flow strip is 107TCID50/ml for 1766PCV2,106TCID50/ml for 1767PCV2. The limit concentration of the 8A12-marked lateral flow strips is 108TCID50/ml for 1767PCV2. The expiration of the lateral flow strips is 12 monthes at 4℃or at room-temperature.
引文
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