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猪丁型冠状病毒检测方法的建立及初步应用

标题: 猪丁型冠状病毒检测方法的建立及初步应用
英文标题: Establishment and Preliminary Application of the Detection Method of Porcine Deltaconoravirus
作者: 罗尚星
出版时间: 2018-06-01
所在大学: 河北农业大学
关键词: 猪丁型冠状病毒,M蛋白,诊断方法,ELISA,荧光定量
英文关键词: Porcine deltacoronavirus,M protein,Diagnostic methods,ELISA,Fluorescence quantitativity
论文级别: 硕士
学位: 硕士
导师: 左玉柱
专业: 预防兽医学
主题: 猪丁型冠状病毒检测方法的建立及初步应用
页码: 66
摘要: 猪丁型冠状病毒(Porcine deltacoronavirus,PDCo V)是冠状病毒科(Coronavirinae)δ-冠状病毒属的成员,是近年来发现的呈世界性流行的猪肠道病毒病。其临床症状表现为急性腹泻、呕吐、脱水。该病毒首先于2012年在香港被报道,2014年后,该病相继在美国韩国等国家被报道,之后便以暴发的方式在世界范围内发生与流行,对养猪业的健康发展造成了一定的影响。PDCo V的M基因和N基因比较保守,另外有研究表明,PDCo V与PEDV,TGEV的M蛋白抗原间没有交叉性反应性。因此,可以利用PDCo V的M基因和N基因构建检测方法,将为PDCo V防控提供一定的技术支持。本实验室进行了一系列研究:(1)根据Gen Bank中已发表的猪丁型冠状病毒M全基因序列进行引物设计并合成,M基因经PCR扩增,克隆测序鉴定后的质粒命名为PDCo V-M。根据p ET32a载体和M蛋白的特点进行表达引物设计,将p ET32a载体与从克隆质粒PDCo V-M中扩增的M基因连接转化构建重组表达质粒p ET32a-M并进行诱导,SDS-PAGE检测有与预期相合的50 ku左右的蛋白大量表达且蛋白主要存在于包涵体中,利用His标签对p ET32a-M蛋白进行纯化,Western-blotting显示p ET32a-M蛋白的反应原性较好。(2)利用纯化的M重组蛋白作为抗原进行了PDCo V M蛋白Ig G抗体的间接ELISA检测方法的构建,对反应条件优化和选择的结果显示M重组蛋白的适宜反应浓度为4μg/m L,37℃恒温反应2小时,4℃放置8~12 h(或过夜)包被结果更好;采用PBST工作液溶解的脱脂奶粉(5%)进行封闭效果最好,37℃恒温反应1小时;血清和酶标抗体的最适稀释度分别为1∶100和1∶5000,两者均37℃恒温反应1小时;TMB暗处反应20 min最佳。且此方法重复性试验(批内和批间)显示变异系数均不超过10%,与CSFV、PRRSV、PEDV、TGEV阳性血清间不存在抗原交叉反应,具有较高的重复性与特异性,为PDCo V的大量检测提供了一种适于操作的ELISA诊断方法。(3)利用纯化的M重组蛋白为抗原进行了PDCo V M蛋白Ig A抗体的间接ELISA检测方法的构建,对反应条件优化和选择的结果显示M重组蛋白的适宜反应浓度为1.6μg/m L,4℃放置8~12 h(或过夜)包被结果更好;采用PBST工作液溶解的脱脂奶粉(5%)进行封闭杂蛋白的效果最好,37℃恒温反应1小时;初乳和酶标抗体的最适稀释度分别为1∶80和1∶50000,两者均37℃恒温反应1小时;TMB暗处反应15 min最佳。且此方法重复性试验(批内和批间)显示变异系数不超过10%,与CSFV、PRRSV、PEDV、TGEV阳性初乳间不存在抗原交叉反应,具有较高的重复性与特异性,为PDCo V的早期检测提供了一种简便的ELISA诊断方法。(4)以10倍梯度稀释的纯化重组PDCo V-M质粒作为标准品模板,建立了一种检测PDCo V的SYBR Green I荧光定量PCR方法。结果显示,本试验建立的检测方法在101拷贝·μL-1至107拷贝·μL-1之间线性关系均较好,相关系数R^2为1.00,扩增效率E在99%以上,检测灵敏度为53拷贝·μL-1,且与PEDV、TGEV、PRV、PCV2病毒的DNA均无交叉性反应,特异性较高;组内变异系数小于1.52%,组间变异系数小于3.15%,重复性较好,为PDCo V的诊断及分子流行病学调查提供了一种快速、定量检测方法。(5)通过Gen Bank登录的PDCo V N基因的序列设计了一组LAMP引物,所用的反应模板为p MD19T-N重组质粒,以羟基萘酚蓝为显色剂,通过对反应温度,反应物浓度等进行优化,构建了一种可直接观察结果的检测PDCo V的LAMP检测方法,并对其进行了特异性试验和灵敏度检测。结果表明,该方法特异性较好,与PEDV、TGEV、PRV、PCV2的DNA间均不存在交叉性反应;该方法灵敏度较好,可检测到的最低模板量为51拷贝·μL-1,为PDCo V的临床检测提供了一种新的方法。
英文摘要: Porcine deltacoronavirus(PDCo V)is a member of Coronavirinae of δ-coronavirus genus,which is a worldwide epidemic of swine enterovirus in recent years.The clinical symptoms of watery diarrhea,vomiting,dehydration,and the virus was reported in the first place in Hong Kong in 2012.After 2014,the disease has been reported in South Korea and other countries in the United States.After that,the outbreak and epidemic in the world have caused a certain impact on the healthy development of the pig industry.The M and N genes of PDCo V are relatively conservative,and other studies have shown that there is no crossover reactivity between PDCo V and PEDV,TGEV's M protein antigen.Therefore,the M gene and N gene of PDCo V can be used to construct the detection method,which will provide certain technical support for the prevention and control of PDCo V.The lab made a series of research:(1)The primers were designed and synthesized according to the M complete gene sequences published in Gen Bank,and the M gene was amplified by PCR,and the plasmid after cloning was identified as PDCo V-M.According to the characteristics of the p ET32 a and M protein primer design,the p ET32 a and M gene which from cloned plasmid PDCo V-M were amplification in connection transformation to construct recombinant expression plasmid p ET32a-M and induction.The SDS-PAGE detects that there is a large amount of expression of protein in the region of 50 ku as expected and the protein is mainly present in the inclusion body,and the protein purified with His tag.The Western blotting shows that the response of the p ET32a-M protein is better.(2)Using recombinant protein as antigen purification of M PDCo V M protein Ig G antibody indirect ELISA detection method of build,optimize the reaction conditions and selection results show that the recombinant protein suitable reaction concentration of 4 μg/m L,37 ℃constant temperature reaction 2 hours,4 placed overnight package is the result is better;5 % skim milk ℃powder of 37 ℃temperature response 1 hour dissolved by PBST is the best sealing effect;The optimal dilution of serum and enzyme antibody was 1:100 and 1:5000,respectively,both 37 ℃temperature response 1 hour;TMB avoid light for 20 min.And this method repeatability test(between batch and batch)shows that the variation coefficient is less than 10%,and there is no cross reaction between CSFV,PRRSV,PEDV,TGEV positive serum,has high repeatability and specificity,to a large number of detection of PDCo V provides a suitable for ELISA diagnostic method of the operation.(3)Using purified PDCo V M recombinant protein as antigen of Ig A antibodies against the construction of the indirect ELISA detection method of the reaction conditions of optimization and selection results show that the recombinant protein of appropriate reaction concentration of 1.6 μg /m L,4 ℃placed overnight package is the result is better;5 % skim milk powder of 37 ℃temperature response 1 hour dissolved by PBST is the best sealing effect;The optimal dilution of serum and enzyme antibody was 1:80 and 1:50000,respectively,both 37 temperature ℃ response 1 hour;TMB avoid light for 15 min.And this method repeatability test(between batch and batch)shows the variation coefficient is less than 10%,and there is no cross reaction between CSFV,PRRSV,PEDV,TGEV positive colostrum,has high repeatability and specificity,to the early detection of PDCo V provides an easy method of ELISA diagnosis.(4)PDCo V-M plasmid was purified by 10 times gradient dilution as the standard template,and a SYBR Green I fluorescent quantitative PCR method was established to detect PDCo V.The results show that the test method established in this experiment has a good linear relationship in 101 copies·μL-1~107 copies·μL-1,the correlation coefficient R^2 is 1.00,E amplification efficiency over 99%,the detection sensitivity of 53 copies·μL-1,and there is no cross reaction between PEDV,TGEV,PRV,PCV2 virus DNA,specificity is higher;The coefficient of variation within the group was less than 1.52%,the variation coefficient of the group was less than 3.15%,and the repeatability was better,which provided a rapid and quantitative detection method for the diagnosis and molecular epidemiological investigation of PDCo V.(5)The sequence of the PDCo V N gene,which was registered through Gen Bank,designed a group of LAMP primers.The reaction template used was p MD19T-N recombinant plasmid,and the reaction temperature,reactant concentration and the like were optimized.A visual inspection method for detection of PDCo V was constructed,and a specific test and sensitivity test were carried out.The results show that the method is very specific,and there is no cross reaction between PEDV,TGEV,PRV,and PCV2 virus DNA;The sensitivity of this method is good,and the minimum number of templates can be detected to 51 copies·μL-1,which provides a new method for the clinical detection of PDCo V.