猪巨细胞病毒快速检测方法的建立及河南猪群流行病学调查
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摘要
猪巨细胞病毒病是由致猪发病的猪巨细胞病毒(Porcine Cytomegalovirus, PCMV)引起的种条件性传染病。长期以来由于对该病研究不够深入,其对猪群的危害性没有得到足够关注。但近年来研究发现该病毒主要在猪肺泡巨噬细胞中增殖破坏机体免疫系统,造成机体感染后抵抗力降低,易并发和继发其他病原体(如猪蓝耳病)感染,PCMV对养猪业的危害逐渐引起了行业人士的高度重视。因此,开展该病的快速诊断方法研究和流行病学调查,对降低该病对养猪业的危害具有重要意义。
本研究应用基因工程技术对PCMV的DNA聚合酶基因特异性片段进行克隆和序列测定；大量比对GenBank中PCMV DNA聚合酶基因序列,设计PCR和荧光定量PCR(FQ-PCR)特异性引物和TaqMan荧光探针,优化反应条件,建立PCMVPCR和FQ-PCR检测方法并对该方法的敏感性、特异性和重复性进行验证,对疑似PCMV感染的临床样品采采用FQ-PCR方法进行应用检测,同时与常规PCR方法进行对比实验,对检测结果为PCMV阳性猪的不同组织器官和血清进行病毒含量测定；对2009年～2011年河南省部分猪群进行了PCMV感染和混合感染的流行病学调查。
结果显示：成功对PCMV的DNA聚合酶基因进行了克隆和序列测定,该基因与GenBank中登录的PCMV DNA聚合酶基因的核苷酸同源性达99.3%；成功建立了PCMV PCR方法和FQ-PCR方法,建立的PCR方法特异性强,敏感性高,重复性好,最低检出量为100拷贝/μL。建立的FQ-PCR方法的标准曲线循环阈值与模板浓度有良好的线性关系,相关系数为0,998；FQ-PCR方法的检测灵敏度可达1拷贝/μL,是常规PCR的100倍；特异性高,对pGEM-T/PCMV重组质粒扩增呈现阳性反应曲线,而6个对照病原的扩增曲线均呈现阴性反应；对不同浓度的pGEM-T/PCMV重组质粒分别重复扩增6次,重复结果良好；对15份临床疑似PCMV感染的组织样品的FQ-PCR应用检测,结果有9份样品为阳性,与常规PCR方法检测的阳性符合率为100%。感染PCMV的猪组织器官和血清中均有病毒存在,含量最高的是扁桃体,最低的是小肠。
对2009年～2011年河南省部分猪群进行PCMV感染状况的流行病学调查结果显示：PCMV的感染呈逐年增加趋势,其中2011年猪群的阳性率最高(58.7%),2010年次之(52.6%),2009年猪群的阳性率较低(44.3%)。对不同饲养模式的猪群进行PCMV感染状况进行流行病学调查显示,PCMV在不同的饲养环境中都有感染,其中散养户猪阳性率最高(60.5%),小规模场次之(50.6%),大规模场最低(33.3%)；对不同年龄的猪群进行PCMV感染状况的流行病学调查显示,PCMV在猪群中感染普遍,其中仔猪阳性率最高(65.2%),母猪次之(47.9%),育肥猪最低(37.9%)；对PCMV与其他病原的混合感染状况进行流行病学调查结果显示：PCMV与猪蓝耳病病毒、猪圆环病毒2型和猪伪狂犬病毒存在二重或三重混合感染现象。
本研究成功建立了PCMV的PCR方法和FQ-PCR方法,用FQ-PCR方法对猪的组织器官和血清中PCMV含量进行了定量检测,对2009年～2011年河南省部分猪群PCMV感染和与其他疫病混合感染状况进行了流行病学调查。该研究结果丰富了我国PCMV流行病学调查内容,加深了对我国PCMV感染现状的了解,对我国PCMV的科学防控将具有重要的指导意义。
The Porcine cytomegalovirus disease caused by Porcine cytomegalovirus (PCMV) is a conditional infectious disease. The research of this disease is not deep for a long time, so the danger to the herd does not get enough attention. But recent studies have found that the virus mainly proliferate in porcine alveolar macrophage, destruct the body's immune system, resulting in reduced animal's resistance after infection, which caused to complicated and secondary infection with other pathogens (such as PRRSV), the harm of Porcine cytomegalovirus to the pig industry gradually attracted attention of industry professional. Therefore, the study on rapid diagnostic method and the epidemiological investigation of the disease will have great significance to reduce the harm of the pig industry.
In this study, DNA polymerase gene of PCMV was cloned and determined by genetic engineering technology, and compared a lot of PCMV DNA polymerase gene sequences in GenBank, then specific primers and TaqMan fluorescent probes to establish PCR and real-time PCR (FQ-PCR) were designed and synthesized, TaqMan fluorescent quantitative real-time PCR (FQ-PCR) to detect PCMV after optimizing the reaction conditions was established, and the sensitivity, specificity and reproducibility of PCR and FQ-PCR were carried out; The suspected samples in clinical were detected by using FQ-PCR method compared with the ordinary PCR and the viral content of the different tissues organs and serum in the porcine cytomegalovirus-positive pigs were quantified; An epidemiological survey of the porcine cytomegalovirus infections and mixed infection in regions of Henan Province was conducted from2009to2011.
The results indicated that the DNA polymerase gene of PCMV was successfully cloned and determined, compared with landing other PCMV polumerase gene, the nucleotide homology was99.3%. Besides PCR and FQ-PCR methods for testing PCMV were successfully established, which had high specificity, high sensitivity and good repeatability. The minimum detection limit of100copies/μL. There was a good linear relationship between the cycle threshold of the standard curve and template concentration in the established FQ-PCR method, and the correlation coefficient was0.998; the detection sensitivity of FQ-PCR method was up to1copy/μL, which was100times than conventional PCR; The specificity of the method was high, and it rendered positive response curve on amplification of pGEM-T/PCMV recombinant plasmid, while negative reaction curves on the amplification of6control pathogens; With6times repeated amplification of different concentrations pGEM-T/PCMV recombinant plasmids, respectively, the results were well. We applied the method to detect15copies of histopathological materials with clinically suspected PCMV infection, in which,9samples were positive, the positive coincidence rate was100%for detection with conventional PCR method. Different internal tissues organs and serum for PCMV infection positive pigs have virus. That is the highest PCMV amount of the internal tissues organs in pigs was tonsils, and the lowest was small intestine.
An epidemiological survey of PCMV infection status in swinery was conducted from2009to2011. The results indicated that the infection of porcine cytomegalovirus was increasing year by year, and the positive rate was highest in2011(58.7%), followed in2010(52.6%), and the lowest rate was in2009(44.3%). At the same time, the results of epidemiological investigation on PCMV infection were various in the different scale farms.Firstly,the positive rate was the most highest in the pigs of scatter-feed (60.5%), secondly, in small-scale field(50.5%), thirdly, in large-scale field(33.3%). The investigation of epidemiological investigation on PCMV infection in different ages showed that porcine cytomegalovirus infection was common in all ages of pigs. Piglets(65.2%) was one of the highest positive rate, then sows(47.9%), and then fattening pigs(37.9%). According to the epidemiological investigation, there were the evidence of duplicate infection(PCMV+PRRSV) and ternary infection(PCMV+PRV+PCⅦ)
In this study, the PCR and FQ-PCR detection method of PCMV was successfully established, The content of PCMV in different tissues organs and serum was quantified, an epidemiological survey of PCMV infection in regions of Henan Province, and mixed infection with other pathogens among swinery was conducted by using the FQ-PCR method.The study enriched the content of PCMV epidemiological investigation, depened the understanding of the status of China's PCMV infection, and will have important guiding significance in scientific control and prevention of the PCMV.
本研究应用基因工程技术对PCMV的DNA聚合酶基因特异性片段进行克隆和序列测定；大量比对GenBank中PCMV DNA聚合酶基因序列,设计PCR和荧光定量PCR(FQ-PCR)特异性引物和TaqMan荧光探针,优化反应条件,建立PCMVPCR和FQ-PCR检测方法并对该方法的敏感性、特异性和重复性进行验证,对疑似PCMV感染的临床样品采采用FQ-PCR方法进行应用检测,同时与常规PCR方法进行对比实验,对检测结果为PCMV阳性猪的不同组织器官和血清进行病毒含量测定；对2009年～2011年河南省部分猪群进行了PCMV感染和混合感染的流行病学调查。
结果显示：成功对PCMV的DNA聚合酶基因进行了克隆和序列测定,该基因与GenBank中登录的PCMV DNA聚合酶基因的核苷酸同源性达99.3%；成功建立了PCMV PCR方法和FQ-PCR方法,建立的PCR方法特异性强,敏感性高,重复性好,最低检出量为100拷贝/μL。建立的FQ-PCR方法的标准曲线循环阈值与模板浓度有良好的线性关系,相关系数为0,998；FQ-PCR方法的检测灵敏度可达1拷贝/μL,是常规PCR的100倍；特异性高,对pGEM-T/PCMV重组质粒扩增呈现阳性反应曲线,而6个对照病原的扩增曲线均呈现阴性反应；对不同浓度的pGEM-T/PCMV重组质粒分别重复扩增6次,重复结果良好；对15份临床疑似PCMV感染的组织样品的FQ-PCR应用检测,结果有9份样品为阳性,与常规PCR方法检测的阳性符合率为100%。感染PCMV的猪组织器官和血清中均有病毒存在,含量最高的是扁桃体,最低的是小肠。
对2009年～2011年河南省部分猪群进行PCMV感染状况的流行病学调查结果显示：PCMV的感染呈逐年增加趋势,其中2011年猪群的阳性率最高(58.7%),2010年次之(52.6%),2009年猪群的阳性率较低(44.3%)。对不同饲养模式的猪群进行PCMV感染状况进行流行病学调查显示,PCMV在不同的饲养环境中都有感染,其中散养户猪阳性率最高(60.5%),小规模场次之(50.6%),大规模场最低(33.3%)；对不同年龄的猪群进行PCMV感染状况的流行病学调查显示,PCMV在猪群中感染普遍,其中仔猪阳性率最高(65.2%),母猪次之(47.9%),育肥猪最低(37.9%)；对PCMV与其他病原的混合感染状况进行流行病学调查结果显示：PCMV与猪蓝耳病病毒、猪圆环病毒2型和猪伪狂犬病毒存在二重或三重混合感染现象。
本研究成功建立了PCMV的PCR方法和FQ-PCR方法,用FQ-PCR方法对猪的组织器官和血清中PCMV含量进行了定量检测,对2009年～2011年河南省部分猪群PCMV感染和与其他疫病混合感染状况进行了流行病学调查。该研究结果丰富了我国PCMV流行病学调查内容,加深了对我国PCMV感染现状的了解,对我国PCMV的科学防控将具有重要的指导意义。
The Porcine cytomegalovirus disease caused by Porcine cytomegalovirus (PCMV) is a conditional infectious disease. The research of this disease is not deep for a long time, so the danger to the herd does not get enough attention. But recent studies have found that the virus mainly proliferate in porcine alveolar macrophage, destruct the body's immune system, resulting in reduced animal's resistance after infection, which caused to complicated and secondary infection with other pathogens (such as PRRSV), the harm of Porcine cytomegalovirus to the pig industry gradually attracted attention of industry professional. Therefore, the study on rapid diagnostic method and the epidemiological investigation of the disease will have great significance to reduce the harm of the pig industry.
In this study, DNA polymerase gene of PCMV was cloned and determined by genetic engineering technology, and compared a lot of PCMV DNA polymerase gene sequences in GenBank, then specific primers and TaqMan fluorescent probes to establish PCR and real-time PCR (FQ-PCR) were designed and synthesized, TaqMan fluorescent quantitative real-time PCR (FQ-PCR) to detect PCMV after optimizing the reaction conditions was established, and the sensitivity, specificity and reproducibility of PCR and FQ-PCR were carried out; The suspected samples in clinical were detected by using FQ-PCR method compared with the ordinary PCR and the viral content of the different tissues organs and serum in the porcine cytomegalovirus-positive pigs were quantified; An epidemiological survey of the porcine cytomegalovirus infections and mixed infection in regions of Henan Province was conducted from2009to2011.
The results indicated that the DNA polymerase gene of PCMV was successfully cloned and determined, compared with landing other PCMV polumerase gene, the nucleotide homology was99.3%. Besides PCR and FQ-PCR methods for testing PCMV were successfully established, which had high specificity, high sensitivity and good repeatability. The minimum detection limit of100copies/μL. There was a good linear relationship between the cycle threshold of the standard curve and template concentration in the established FQ-PCR method, and the correlation coefficient was0.998; the detection sensitivity of FQ-PCR method was up to1copy/μL, which was100times than conventional PCR; The specificity of the method was high, and it rendered positive response curve on amplification of pGEM-T/PCMV recombinant plasmid, while negative reaction curves on the amplification of6control pathogens; With6times repeated amplification of different concentrations pGEM-T/PCMV recombinant plasmids, respectively, the results were well. We applied the method to detect15copies of histopathological materials with clinically suspected PCMV infection, in which,9samples were positive, the positive coincidence rate was100%for detection with conventional PCR method. Different internal tissues organs and serum for PCMV infection positive pigs have virus. That is the highest PCMV amount of the internal tissues organs in pigs was tonsils, and the lowest was small intestine.
An epidemiological survey of PCMV infection status in swinery was conducted from2009to2011. The results indicated that the infection of porcine cytomegalovirus was increasing year by year, and the positive rate was highest in2011(58.7%), followed in2010(52.6%), and the lowest rate was in2009(44.3%). At the same time, the results of epidemiological investigation on PCMV infection were various in the different scale farms.Firstly,the positive rate was the most highest in the pigs of scatter-feed (60.5%), secondly, in small-scale field(50.5%), thirdly, in large-scale field(33.3%). The investigation of epidemiological investigation on PCMV infection in different ages showed that porcine cytomegalovirus infection was common in all ages of pigs. Piglets(65.2%) was one of the highest positive rate, then sows(47.9%), and then fattening pigs(37.9%). According to the epidemiological investigation, there were the evidence of duplicate infection(PCMV+PRRSV) and ternary infection(PCMV+PRV+PCⅦ)
In this study, the PCR and FQ-PCR detection method of PCMV was successfully established, The content of PCMV in different tissues organs and serum was quantified, an epidemiological survey of PCMV infection in regions of Henan Province, and mixed infection with other pathogens among swinery was conducted by using the FQ-PCR method.The study enriched the content of PCMV epidemiological investigation, depened the understanding of the status of China's PCMV infection, and will have important guiding significance in scientific control and prevention of the PCMV.
引文
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