羊传染性脓疱病毒FIL基因克隆表达及分子生物学检测方法的建立与初步应用
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摘要
羊传染性脓疱病毒(Contagious Ecthyma Virus,简称CEV)是痘病毒科、副痘病毒属的双股DNA病毒,主要危害3-6月龄羔羊,引起急性、高度接触性的传染病——羊传染性脓疱。该病毒通过直接接触传染,在感染动物的口腔、唇、舌、鼻孔周围、乳房等部位的黏膜与皮肤形成丘疹、脓疱、溃疡和疣状痂皮。该病常呈群发性流行,传染性强,发病率高,继发感染的死亡率可达50%。吉林省主要养羊地区均有该病发生。该病是人兽共患传染病,给养羊业带来了巨大的经济损失。对人类的健康也构成威胁。
F1L基因(ORF059)长为1011bp,编码39kDa蛋白,位于病毒基因组物理图谱的中央,是一个完整的开放阅读框,高度保守。39kDa蛋白在病毒的成熟过程中起着重要的作用。本研究选择病毒F1L基因为研究对象,通过对羊传染性脓疱病毒分离株F1L基因进行克隆及同源性分析,了解与其他毒株间基因序列差异,并选择保守基因片段作为分子生物学诊断的目的基因,建立新的诊断方法。为有效预防和控制羊传染性脓疱病毒传播,及时的作出准确诊断,提供科学依据。本研究共分为以下五个部分：
1.羊传染性脓疱病毒的分离与鉴定：从吉林省松原市某一发病的羔羊群中,采取羊嘴唇等部位病变组织,处理后感染实验室自制的新生犊牛肾原代细胞,经细胞连续传代,细胞出现稳定病变。对该病毒进行了TCID50测定、病毒核酸类型鉴定试验、理化培养特性研究、电镜观察、动物回归试验及PCR检测,鉴定该株病毒为羊传染性脓疱病毒,命名为JLSY’株。
2.羊传染性脓疱病毒吉林分离株F1L基因的克隆与同源性分析：常规方法制备新生犊牛肾原代细胞,接种本实验室自行分离的羊传染性脓疱病毒JLSY株,观察细胞病变,证明细胞感染病毒,收集病毒液。通过参考GenBank中羊传染性脓疱病毒的F1L基因序列设计引物,应用PCR技术扩增出羊传染性脓疱病毒JLSY株的F1L基因片段,与pMD18-T载体连接,转化至E.coli JM109中,阳性质粒通过PCR扩增和酶切鉴定后,选择2株含阳性质粒菌进行基因测序,并将F1L基因序列与几个国内外参考毒株的序列进行同源性比较和分析。结果显示,羊传染性脓疱病毒JLSY株与OV/Torino株同源性最低达97.6%,与Jilin株同源性最高达98.8%。说明羊传染性脓疱病毒JLSY株F1L基因与国内其他参考毒株之间差异不大,是病毒的保守的基因,可以选择该保守基因片段作为分子生物学诊断的目的基因。
3.羊传染性脓疱病毒JLSY株F1L基因原核表达及蛋白纯化：设计含有特定酶切位点的引物,扩增出的F1L基因,用EcoR I和Xho I双酶切回收的PCR产物和pET28a载体,分别回收目的片段和载体片段进行连接,构建质粒pET28a-F1L、pET28a-F1L2,然后转化TOP10感受态细胞。提取测序正确的阳性克隆质粒,并转化入BL21表达菌株中。接种于LB培养液中培养,加入IPTG以诱导蛋白表达。表达产物经聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,出现预期目的蛋白条带,可溶性F1L蛋白经亲和层析纯化,进行浓缩,蛋白浓度为0.6mg/mL。
4.羊传染性脓疱病毒环介导等温扩增(LAMP)检测方法的建立及初步应用：参照GenBank中羊传染性脓疱病毒基因序列,针对病毒F1L基因保守区域设计扩增引物,通过对实验条件的优化,建立了基于LAMP技术的CEV快速检测方法,进行了该方法敏感性、特异性、重复性试验,采集临床样品进行初步应用。结果显示,阳性样品扩增产物在琼脂糖凝胶电泳检测中呈特征性梯状条带,阳性反应管加入荧光染料后变成绿色；对CEV的最低检出量为102拷贝／反应,比常规PCR方法高100倍；本方法检测山羊痘病毒、鸡痘病毒、口蹄疫病毒O型、口蹄疫病毒亚洲Ⅰ型病毒为阴性；相同样品进行10次检测,所得检测结果一致。由此说明,本研究建立的CEV LAMP检测方法敏感性高、特异性强、稳定性好。在LAMP反应体系中,加入1μL10×SYBR Green I,可进行实时荧光LAMP检测和定量实验。对30份临床样品进行检测,LAMP检测方法与常规PCR方法检测结果一致。本研究建立的LAMP检测方法,为CEV的快速检测和羊传染性脓疱病的临床诊断提供了一种方便快捷的方法。
5.羊传染性脓疱病毒变性高效液相色谱检测方法的建立及初步应用：将PCR技术和变性高效液相色谱技术(DHPLC)相结合,参考GenBank中羊传染性脓疱病毒的F1L基因序列,应用Primer Premier5.0软件设计引物,应用PCR技术特异性地扩增出羊传染性脓疱病毒JLSY株的F1L基因片段,对PCR扩增产物使用变性高效液相色谱仪进行检测与分析,建立检测方法。进行了敏感性试验、特异性试验、稳定性试验和临床应用试验。结果表明：PCR-DHPLC检测方法能检测到最低拷贝数为103,其敏感性比常规PCR电泳法高100倍；对CEV基因组DNA和质粒标准品的检测结果为阳性,而对其它常见动物病毒基因组DNA的检测结果为阴性,表明本检测方法特异性较好。使用PCR-DHPLC方法和PCR电泳法对30份临床样本进行检测,两种方法阳性样品的检出符合率为100%。本研究建立的PCR-DHPLC检测方法敏感性更高,特异性更强,稳定性更好,可用于检测羊传染性脓疱病毒,为羊传染性脓疱病诊断提供是一种新方法。
Contagious Ecthyma virus (CEV) is Poxviridae which is Parapoxvirus with double stranded DNA virus. CEV is infectious disease which mainly causes proliferative lesions of infected skin and mucosa of lambs aged3-6months old. The disease spread by physical contact which is characterized by the formation of papules, vesicles, ulcers or that progress into heavy scabs on oral cavities, lips, tongues, around nostrils, breast. The disease usually spread group-occurring and the infectivity and morbidity of which is very high inside which the secondary mortality rate is up to50%. The virus spread through all of the main sheep-breeding area of Jilin Province. The disease is zoonosis, it brings huge economic loss and health risk to sheep-breeding and human beings.
F1L gene is a highly conserved open reading frame with1011bp long and39kDa proteins which located in the middle of the virus genome physical map.39kDa proteins plays an important role during the ripening stages of virus. This study choose virus F1L gene as the research object,, which investigating the differences of gene sequence between this strain with another one through cloning and homologic analysis of the F1L gene of CEV isolated strain. Choosing the censervative gene fragment as the target gene for molecular biological diagnostic and exploring a practical method for restrain the spreading of CEV which could brings the important basis-and establishing the diagnosis timely. The study is divided into the following five parts.
1. Isolation and identification of CEV:Using lesion tissues near by lamb's lips which among a flock of disease lambs in Songyuan of Jilin province, Infecting newborn calf primary kidney cells self-made by lab after treatment, cells appears stable lesions by serial passage of cells. The study appraised the strain is contagious ecthyma virus and named it JLSY by the TCID50determination of the virus, type of evaluation test on virus nucleic acid, characteristic study on physical and chemical cultured, electron microscopic observation, animal regression experiment and PCR detection.
2. Cloning and homologic analysis of the F1L gene of CEV from Jilin isolate:prepared newborn calf primary kidney cells by conventional methods, inoculated CEV JLSY which screened by our laboratory, observing cytopathic effect to prove Cells infected with virus, collect the virus liquid. According to the DNA sequence of the FIL gene which was published on GenBank, we designed one pair of primers to amplify F1L gene of JLSY by PCR. Link the fragment vector with pMD18-T, transformed into E.coli JM109, choose2strain virus which contain positive plasmid to keep gene sequencing before positive plasmid amplifying PCR and enzyme digestion. The amplified positive production was sequenced and compared with the sequences of several reference strains isolated in domestic. The results showed that the lowest homology with F1L gene of JLSY strains was97.6%of Strain OV/Torino, and the highest homology of Jilin strain was up to98.8%. There was little difference of F1L gene between JLSY strain and other reference strains isolated from different species and different areas in domestic. The analysis result of homology suggested that the F1L gene can be used as the target genes of the genetically engineered vaccine.
3. Construction, expression and purification of CEV JLSY F1L expression vector: Designing primers which containing specific enzyme sites, amplified F1L gene, using PCR products and pET28a vector recovered by Double-enzyme Digestion of EcoRI and XhoI. Combing target fragment and vector fragment which were recovered respectively, constructing pET28a-F1L and pET28a-F1L2plasmid, and then transforming TOP10competent cells. Extracting right sequenced posrtive cloning plasmid and transforming it into BL21expression strain. Inoculating in LB culture medium in which we mix IPTF to lead the expression of protein. The expression produets detecting by SDS-PAGE, appearing protein band which is our expected goal. Concentrating soluble F1L protein which was purified by affinity chromatography, the protein concentration is0.6mg/mL.
4. Establishment and preliminary application of CEV loop-mediated isothermal amplification diagnostic Methods:According to the CEV gene sequence published in GenBank, targeting a highly conserved region of the F1L gene has been developed to diagnose CEV. Sensitivity test, specificity test and replicate test of this method were promoted and then collecting clinical samples to develop preliminary application. The assay produces a ladder-like pattern of products on an agarose gel. Postive reaction tube turns green when the flurescent dye is added. The sensitivity of the LAMP assay, which was determined to be102copies of the standard plasmid, was100fold higher than PCR; furthermore, the method detection of capripox virus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I is negative;the same samples were detected for10times and the results were the same. It shows that the CEV LAMP detection method established by our study was much more sensitive specific and stable. The LAMP assay with lμL10×SYBR Green I may carry out the real-time LAMP detection and semi-quantitation experiment.30clinical samples were tested using PCR and LAMP assay, and the LAMP test results were consistent with PCR methods. The LAMP assay allows easy and rapid detection of infection with CEV and is especially applicable in a resource-limited situation.
5. Establishment and preliminary application of CEV (PCR-DHPLC) diagnostic Methods: According to the gene sequence of the CEV F1L strain which published in GenBank, combining PCR with denaturing high performance liquid chromatography, designing primers with Primer Premier5.0, we designed one pair of primers to amplify F1L gene of JLSY by PCR. Analysed and detected by denaturing high performance liquid chromatography, established the detection method with PCR amplification. Sensitivity test, specificity test, stability test and clinical application test showed that the lowest copy number found by PCR-DHPLC detection method is103. The sensitivity is100higher than regular PCR electrophoresis. The specificity of our detection method is very well, which because the result of other animal virus gene DNA detection is negative and the result of CEV gene DNA with standard plasmid detection is positive. The coincidence of positive sample is100%by which using PCR-DHPLC and PCR electrophoresis detection method to detect the30clinical samples. The PCR-DHPLC detection method established by this study was much more sensitive specific and stable which could be an efficient new one for detecting the CEV.
F1L基因(ORF059)长为1011bp,编码39kDa蛋白,位于病毒基因组物理图谱的中央,是一个完整的开放阅读框,高度保守。39kDa蛋白在病毒的成熟过程中起着重要的作用。本研究选择病毒F1L基因为研究对象,通过对羊传染性脓疱病毒分离株F1L基因进行克隆及同源性分析,了解与其他毒株间基因序列差异,并选择保守基因片段作为分子生物学诊断的目的基因,建立新的诊断方法。为有效预防和控制羊传染性脓疱病毒传播,及时的作出准确诊断,提供科学依据。本研究共分为以下五个部分：
1.羊传染性脓疱病毒的分离与鉴定：从吉林省松原市某一发病的羔羊群中,采取羊嘴唇等部位病变组织,处理后感染实验室自制的新生犊牛肾原代细胞,经细胞连续传代,细胞出现稳定病变。对该病毒进行了TCID50测定、病毒核酸类型鉴定试验、理化培养特性研究、电镜观察、动物回归试验及PCR检测,鉴定该株病毒为羊传染性脓疱病毒,命名为JLSY’株。
2.羊传染性脓疱病毒吉林分离株F1L基因的克隆与同源性分析：常规方法制备新生犊牛肾原代细胞,接种本实验室自行分离的羊传染性脓疱病毒JLSY株,观察细胞病变,证明细胞感染病毒,收集病毒液。通过参考GenBank中羊传染性脓疱病毒的F1L基因序列设计引物,应用PCR技术扩增出羊传染性脓疱病毒JLSY株的F1L基因片段,与pMD18-T载体连接,转化至E.coli JM109中,阳性质粒通过PCR扩增和酶切鉴定后,选择2株含阳性质粒菌进行基因测序,并将F1L基因序列与几个国内外参考毒株的序列进行同源性比较和分析。结果显示,羊传染性脓疱病毒JLSY株与OV/Torino株同源性最低达97.6%,与Jilin株同源性最高达98.8%。说明羊传染性脓疱病毒JLSY株F1L基因与国内其他参考毒株之间差异不大,是病毒的保守的基因,可以选择该保守基因片段作为分子生物学诊断的目的基因。
3.羊传染性脓疱病毒JLSY株F1L基因原核表达及蛋白纯化：设计含有特定酶切位点的引物,扩增出的F1L基因,用EcoR I和Xho I双酶切回收的PCR产物和pET28a载体,分别回收目的片段和载体片段进行连接,构建质粒pET28a-F1L、pET28a-F1L2,然后转化TOP10感受态细胞。提取测序正确的阳性克隆质粒,并转化入BL21表达菌株中。接种于LB培养液中培养,加入IPTG以诱导蛋白表达。表达产物经聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,出现预期目的蛋白条带,可溶性F1L蛋白经亲和层析纯化,进行浓缩,蛋白浓度为0.6mg/mL。
4.羊传染性脓疱病毒环介导等温扩增(LAMP)检测方法的建立及初步应用：参照GenBank中羊传染性脓疱病毒基因序列,针对病毒F1L基因保守区域设计扩增引物,通过对实验条件的优化,建立了基于LAMP技术的CEV快速检测方法,进行了该方法敏感性、特异性、重复性试验,采集临床样品进行初步应用。结果显示,阳性样品扩增产物在琼脂糖凝胶电泳检测中呈特征性梯状条带,阳性反应管加入荧光染料后变成绿色；对CEV的最低检出量为102拷贝／反应,比常规PCR方法高100倍；本方法检测山羊痘病毒、鸡痘病毒、口蹄疫病毒O型、口蹄疫病毒亚洲Ⅰ型病毒为阴性；相同样品进行10次检测,所得检测结果一致。由此说明,本研究建立的CEV LAMP检测方法敏感性高、特异性强、稳定性好。在LAMP反应体系中,加入1μL10×SYBR Green I,可进行实时荧光LAMP检测和定量实验。对30份临床样品进行检测,LAMP检测方法与常规PCR方法检测结果一致。本研究建立的LAMP检测方法,为CEV的快速检测和羊传染性脓疱病的临床诊断提供了一种方便快捷的方法。
5.羊传染性脓疱病毒变性高效液相色谱检测方法的建立及初步应用：将PCR技术和变性高效液相色谱技术(DHPLC)相结合,参考GenBank中羊传染性脓疱病毒的F1L基因序列,应用Primer Premier5.0软件设计引物,应用PCR技术特异性地扩增出羊传染性脓疱病毒JLSY株的F1L基因片段,对PCR扩增产物使用变性高效液相色谱仪进行检测与分析,建立检测方法。进行了敏感性试验、特异性试验、稳定性试验和临床应用试验。结果表明：PCR-DHPLC检测方法能检测到最低拷贝数为103,其敏感性比常规PCR电泳法高100倍；对CEV基因组DNA和质粒标准品的检测结果为阳性,而对其它常见动物病毒基因组DNA的检测结果为阴性,表明本检测方法特异性较好。使用PCR-DHPLC方法和PCR电泳法对30份临床样本进行检测,两种方法阳性样品的检出符合率为100%。本研究建立的PCR-DHPLC检测方法敏感性更高,特异性更强,稳定性更好,可用于检测羊传染性脓疱病毒,为羊传染性脓疱病诊断提供是一种新方法。
Contagious Ecthyma virus (CEV) is Poxviridae which is Parapoxvirus with double stranded DNA virus. CEV is infectious disease which mainly causes proliferative lesions of infected skin and mucosa of lambs aged3-6months old. The disease spread by physical contact which is characterized by the formation of papules, vesicles, ulcers or that progress into heavy scabs on oral cavities, lips, tongues, around nostrils, breast. The disease usually spread group-occurring and the infectivity and morbidity of which is very high inside which the secondary mortality rate is up to50%. The virus spread through all of the main sheep-breeding area of Jilin Province. The disease is zoonosis, it brings huge economic loss and health risk to sheep-breeding and human beings.
F1L gene is a highly conserved open reading frame with1011bp long and39kDa proteins which located in the middle of the virus genome physical map.39kDa proteins plays an important role during the ripening stages of virus. This study choose virus F1L gene as the research object,, which investigating the differences of gene sequence between this strain with another one through cloning and homologic analysis of the F1L gene of CEV isolated strain. Choosing the censervative gene fragment as the target gene for molecular biological diagnostic and exploring a practical method for restrain the spreading of CEV which could brings the important basis-and establishing the diagnosis timely. The study is divided into the following five parts.
1. Isolation and identification of CEV:Using lesion tissues near by lamb's lips which among a flock of disease lambs in Songyuan of Jilin province, Infecting newborn calf primary kidney cells self-made by lab after treatment, cells appears stable lesions by serial passage of cells. The study appraised the strain is contagious ecthyma virus and named it JLSY by the TCID50determination of the virus, type of evaluation test on virus nucleic acid, characteristic study on physical and chemical cultured, electron microscopic observation, animal regression experiment and PCR detection.
2. Cloning and homologic analysis of the F1L gene of CEV from Jilin isolate:prepared newborn calf primary kidney cells by conventional methods, inoculated CEV JLSY which screened by our laboratory, observing cytopathic effect to prove Cells infected with virus, collect the virus liquid. According to the DNA sequence of the FIL gene which was published on GenBank, we designed one pair of primers to amplify F1L gene of JLSY by PCR. Link the fragment vector with pMD18-T, transformed into E.coli JM109, choose2strain virus which contain positive plasmid to keep gene sequencing before positive plasmid amplifying PCR and enzyme digestion. The amplified positive production was sequenced and compared with the sequences of several reference strains isolated in domestic. The results showed that the lowest homology with F1L gene of JLSY strains was97.6%of Strain OV/Torino, and the highest homology of Jilin strain was up to98.8%. There was little difference of F1L gene between JLSY strain and other reference strains isolated from different species and different areas in domestic. The analysis result of homology suggested that the F1L gene can be used as the target genes of the genetically engineered vaccine.
3. Construction, expression and purification of CEV JLSY F1L expression vector: Designing primers which containing specific enzyme sites, amplified F1L gene, using PCR products and pET28a vector recovered by Double-enzyme Digestion of EcoRI and XhoI. Combing target fragment and vector fragment which were recovered respectively, constructing pET28a-F1L and pET28a-F1L2plasmid, and then transforming TOP10competent cells. Extracting right sequenced posrtive cloning plasmid and transforming it into BL21expression strain. Inoculating in LB culture medium in which we mix IPTF to lead the expression of protein. The expression produets detecting by SDS-PAGE, appearing protein band which is our expected goal. Concentrating soluble F1L protein which was purified by affinity chromatography, the protein concentration is0.6mg/mL.
4. Establishment and preliminary application of CEV loop-mediated isothermal amplification diagnostic Methods:According to the CEV gene sequence published in GenBank, targeting a highly conserved region of the F1L gene has been developed to diagnose CEV. Sensitivity test, specificity test and replicate test of this method were promoted and then collecting clinical samples to develop preliminary application. The assay produces a ladder-like pattern of products on an agarose gel. Postive reaction tube turns green when the flurescent dye is added. The sensitivity of the LAMP assay, which was determined to be102copies of the standard plasmid, was100fold higher than PCR; furthermore, the method detection of capripox virus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I is negative;the same samples were detected for10times and the results were the same. It shows that the CEV LAMP detection method established by our study was much more sensitive specific and stable. The LAMP assay with lμL10×SYBR Green I may carry out the real-time LAMP detection and semi-quantitation experiment.30clinical samples were tested using PCR and LAMP assay, and the LAMP test results were consistent with PCR methods. The LAMP assay allows easy and rapid detection of infection with CEV and is especially applicable in a resource-limited situation.
5. Establishment and preliminary application of CEV (PCR-DHPLC) diagnostic Methods: According to the gene sequence of the CEV F1L strain which published in GenBank, combining PCR with denaturing high performance liquid chromatography, designing primers with Primer Premier5.0, we designed one pair of primers to amplify F1L gene of JLSY by PCR. Analysed and detected by denaturing high performance liquid chromatography, established the detection method with PCR amplification. Sensitivity test, specificity test, stability test and clinical application test showed that the lowest copy number found by PCR-DHPLC detection method is103. The sensitivity is100higher than regular PCR electrophoresis. The specificity of our detection method is very well, which because the result of other animal virus gene DNA detection is negative and the result of CEV gene DNA with standard plasmid detection is positive. The coincidence of positive sample is100%by which using PCR-DHPLC and PCR electrophoresis detection method to detect the30clinical samples. The PCR-DHPLC detection method established by this study was much more sensitive specific and stable which could be an efficient new one for detecting the CEV.
引文
[1]苏风鸣.羔羊传染性口膜炎的研究[J].科学研究年报,1964,(3)：54.
[2]邵洪泽,刘云志,程荣华,等.羊传染性脓疱病毒的分离与鉴定[J].吉林畜牧兽医,2006,27(11)：11-12.
[3]赵魁,高丰,贺文琦,等.1株长春地区羊传染性脓疱病毒的分离鉴定及F1L基因的克隆与序列分析[J].中国兽医学报,2010,(7)：931-935.
[4]殷震,刘景华.动物病毒学[M].第二版,北京：科学出版社,1997,977-980.
[5]王光华,陆艳,王戈平,等.羊传染性脓疱诊断方法的建立[J].青海畜牧兽医杂志,2012,05：1-3.
[6]陈光辉.一起羊传染性脓疱的防治[J].甘肃畜牧兽医,2004,(4)：32-33.
[7]周江名,夏刚位.羊传染性脓疱综合防治[J].四川畜牧兽医,2011,(6)：54-55.
[8]闫喜军,卢金明,王艳华.羊传染性脓疱病的治疗[J].养殖技术顾问,2004,(2)：29.
[9]李瑞芳,乔军,张辉,等.羊传染性脓疱病毒新疆流行株F1L基因克隆及表达[J].生物技术,2012,(3)：17-20.
[10]王辉,赵晓军,易建刚,等.羊传染性脓疱病毒内蒙分离株F1L基因的克隆与序列分析[J].中国动物检疫,2007,(6)：26-27.
[11]赖自武,郑建珍,陈家俊.羊传染性脓疱的综合防治[J].云南农业,2005,(5)：14.
[12]张克山,何继军,尚佑军,等.羊传染性脓疱病毒湖北株的鉴定及分子特征分析[J].畜牧兽医学报,2010,(9)：1154-1157.
[13]于恩庶,林继煌,陈观今.中国人兽共患病学(第二版)[M].福州：福建科学技术出版社,1996,753-758.
[14]中国农业科学院哈尔滨兽医研究所.动物传染病学[M].北京：中国农业出版社,1998,363-365.
[15]蔡宝祥.家畜传染病学[M].北京：中国农业出版社,2001,157-159.
[16]蔡宝祥,殷震,谢三星,等.动物传染病诊断学[M].江苏科学技术出版社,1993,187-188.
[17]Straten M van, Friedgut O. Outbreak of contagious ecthyma in carnal in srael[J]. Vet Reco,2001,148:150-151.
[18]Kummeneje K, Krogsrud J. Contagious ecthyma (Orf) in the musk ox [J]. Acta Vet Scand,1978,19:461-462.
[19]Kummeneje K, Krogsrud J. Contagious ecthyma (Orf) in reindeer[J]. Vet Rec,1979,105:60-61.
[20]WilkinsonG T, Prydie J, Scarnell J. Possible"orf'(contagious pustular dermatitis, contagious ecthyma of sheep) infection in the dog[J]. Vet Rec,1970,87:766-767.
[21]Nollens H H, Jacobson E R, Gulland F M D, et al. Pathology and preliminary characterization of a parapoxvirus isolated from a california sea lion (Zalophus californianus)[J]. Journal of Wildlife Diseases,2006,42(1):23-32.
[22]Becher P, Konig M, Muller G, et al. Characterization of sealpoxvirus, a separate member of the parapoxvirus [J].Arch Virol, 2002,147:1133-1140.
[23]王积禄,等.羊传染性口膜炎[J].中国畜牧兽医,1962,(2)：10-11.
[24]陈燕军.羊口疮-病原学及试验病理学研究[J].兽医科技杂志,1982,(6)：2-8.
[25]Groves R W, Wilson-Jones E, MacDonald D M. Human orf and milkers'nodule:a clinicopathologic study[J]. J Amer Acad Dermatol,1991,25:706-711.
[26]Kuhl J T, Huerter C J, Hashish H. A case of human orf contracted from a deer[J]. Cutis.2003,71(4):288-289.
[27]甘肃农业大学主编.家畜传染病学[M].北京：农业出版社,1984,365-367.
[28]Czerny C P, Waldmann R, Scheubeck T. Identification of three distinct antigenic sites in parapoxvirus[J]. Arch Virol,1997, 142:807-821.
[29]A Boxton, et al. Animal microbiology[M].1977,670-691.
[30]Rossi G A. A daptaation of the virus of contagious ecthyma to cellular substrates of avian origin[J]. Vet Ital,1973,24: 218-222.
[31]Robinson A J,Barns G,Fraaer K,et.al. Conservation and Variation in orf virus genores[J]. Virology,1987,157:13-23.
[32]Greig A S. Contagious ecthyma of sheep Ⅱ.In vitro cultivation of the virus[J]. Can J comp Med,1957,21:304-308.
[33]赵立志,薛双虎,吕武夫,等.羊传染性农疱皮炎病毒的细胞培养[J].中国兽医杂志,1991,17(8)：12-13.
[34]何水林,陈杰,蔡玉书.羊口疮病毒的分离鉴定[J].中国兽医科技,2002,9：22-23.
[35]Pye D. Vaccination of sheep with cell culture grown orf virus[J]. Aust Vet J,1990,67:182-186.
[36]Straten M van,Friedgut O. Outbreak of contagious ecthyma in camal in Israel [J]. Vet Reco,2001,148:150-151.
[37]Plowright W, Whitcomb M A, Ferris R D. Studies with a strain of contagious pustular dermatitis virus in tissue culture[J]. Arch Ges virus forsch,1959,9:214-231.
[38]白吉荣,沈正达,王锡祯,等.羊口疮病毒在单层细胞上繁殖特性的研究[J].甘肃农业大学学报,1985,(4)：109-116.
[39]庞理化,程庆华,侯秀芬,等.羊口疮病毒的组织培养初报[J].中国兽医杂志,1982,(3)：66-69.
[40]詹述宣,薛双虎,薛掌林.犊牛睾丸细胞培养羊接触传染性脓疱皮炎病毒的研究[J].兽医科技杂志,1983,(7)：5-7.
[41]Robinson A J, Ellis G, Balassu T C. The genome of orf virus:restrictionendonuclease analysis of viral DNA isolated from lesions of orf in sheep[J]. Arch. Virol,1982,71,43-55.
[42]Gassmann U, Wyler R, Wittek R. Analysis of parapoxvirus genomes[J]. Arch. Virol.,1985,83:17-31.
[43]Cottone R, Biittner M, Bauer B, et al. Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[J]. Virus Res,1998,56:53-67.
[44]Mercer A A, Fraser K, Barns G, et al. The structure and cloning of orf virus DNA[J]. Virology,1987,157:1-12.
[45]Haig D M, Mcinnes C J, Thomson J, et al. The orf virus OV20.OL gene product is involved in interferon resistance and inhibit san interferon 2 inducible, double 2st randed RNA2 dependent kinase[J]. Immunology,1998,93:335-340.
[46]Spehner D, De Carlo S, Drillien R, et al. Appearance of the bona fide spiral tubule of Orf virus is dependent on an ntact 10-kilodalton viral protein[J]. Virology,2004,8085-8093.
[47]Cottone R, But tner M, Mclnnes C J, et al. Orf virus encodes afunctional dU TPase gene[J].General Virology,2002,83: 1043-1048.
[48]Marsland B J, Tisdall D J, Heat h D D, et al. Const ruction of a recombinant orf virus t hat expresses an Echinococcus granulosus vaccine antigen from a novel genomic insertion site[J]. Arch Virol,2003,148 (3):555-562.
[49]Einar G, Torfason, Sigrun. Polymerase chain reaction for laboratory diagnosis of orf virus infection[J]. J Clinic Virol,2002, 24:79-84.
[50]Sullivan J T, Mercer A A, Fleming S B, et al. Identification and characterization of an orf virus homologue of vaccinia virus gene encoding t he major envelop antigen p37k[J].Virology,1994,202:968-973.
[51]Becher P, Konig M, Muller G, et al. Characterization of sealpox virus, a separate member of the parapoxvirus [J]. Arch Vir ol,2002,147:1133-1140.
[52]Wise LM, Veikkola T, Mercer AA, et al. Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1[J]. Proc Natl Acad Sci USA,1999,96:3071-3076.
[53]Seet BT, McCaughan CA, Handel TM, et al. Analysis of an orf virus chemokine-binding protein:Shifting ligand specificiti es among a family of poxvirus viroceptors[J]. PNAS,2003,100 (25):15137-15142.
[54]Haig DM, Mclnnes CJ. Immunity and counter-immunity during infection with the parapoxvirus orf virus[J]. Virus Res,200 2a,88:3-16.
[55]王廷璞,赵菲佚,安建平,等.羊传染性脓疱病毒42K囊膜蛋白基因克隆及表达[J].中国预防兽医学报,2006,28(1)：29-32.
[56]陈燕军.羊口疮的研究—病原学及试验病理学研究[J].兽医科技杂志,1982,6：2-8.
[57]白积荣,沈正达,王锡贞,等.羊口疮病毒形态发生的电镜观察[J].中国兽医科技,1985,(11)：34-36.
[58]Harkness J W, Scott A C, Hebert C N. Electron microscopy in the rapid diagnosis of ORF[J]. British Vet J,1977,133: 81-87.
[59]陈燕军.羊口疮的研究—血清学方法的研究[J].兽医科技杂志,1982,7：14-19.
[60]薛掌林,薛双虎,吕武夫.中和试验对羊传染性脓疱皮炎抗体的检测[J].甘肃畜牧兽医,1990,2：13-14.
[61]Robinson A J, Ellis G,Balassu T C.The genome of orfvirus:Restrictionendonucle seanalys is of viral DNA isolatedfrom lesion so for finsheep[J]. ArchivesofVirology,1982,71:43-55.
[62]王延璞,薛双虎,吕武夫.酶联免疫吸附试验检测CEV[J].甘肃畜牧兽医,1995,25(1)：3-4.
[63]Chin JC, Petersen RK. Comparison of native and subunit antigens as ELISA reagents for thedetection of antibodies against scabby mouth virus[J]. Vet Microbiol,1995,46:327-334.
[64]Chand P, Kitching R P, Black D N. Western blot analysis of virus-specific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep[J]. Epidemiology & Infection,1994,113:377-385.
[65]伍新尧,罗超,等.分子遗传学与基因工程[M].郑州：河南医科大学出版社,1997,157-170.
[66]G H凯勒,M M马纳克.DNA探针技术[M].北京：科学出版社,1992,186-187.
[67]Robinson A J, Ballassu T C,Contagious Pustular dermatitis (Orf) [J]. Vet Bull,1981,51:771-782.
[68]Andrew A Mencr, Kate Fraser, GillianBarns, et al. the structure and cloning of orf viring DNA[J]. virology,1987,157: 11-21.
[69]王廷璞,赵菲佚,孙春香,等.光敏生物素和DIG标记Orf病毒DNA探针的研究[J].生物技术,2002,12(6)：21-22.
[70]李会荣,龚振华,郑增忍.应用PCR检测羊接触CE性皮炎病毒[J].中国兽医科技,2003,33：10-13.
[71]刘云志,邵洪泽,程荣华,等.羊传染性脓疱病毒PCR检测方法的建立及应用[J].吉林畜牧兽医,2007,28(3)：7-9.
[72]Einar G.Torfason, sigrun Gu nadottir.Polymerase chain reaction for laboratory diagnosis of orf virus infections[J]. J chin virol,2002,24:79-84.
[73]Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res. 2000,28:63.
[74]Kurosaki Y, Takada A, Ebihara H, et al. Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification[J]. Journal of virological methods.2007,141:78-83.
[75]Yoshino M, Watari H, Kojima T, et al. Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification[J]. Microbiology and immunology,2009,53:375-383.
[76]Kono T, Savan R, Sakai M, et al. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification[J]. Journal of virological methods,2004,115:59-65.
[77]Aryan E, Makvandi M, Farajzadeh A, et al. A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex[J]. Microbiological Research,2010,165:211-220.
[78]Horibe D, Ochiai T, Shimada H, et al. Rapid detection of metastasis of gastric cancer using reverse transcription loop-mediated isothermal amplification[J]. Int J Cancer.2007 Mar 1,120(5):1063-1069.
[79]Saleh M, Soliman H and El-Matboulicrossref M. Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish[J]. BMC Veterinary Research,2008,4:31.
[80]胡嘉欣,温永俊,霍志云,等.犬瘟热病毒RT-LAMP检测方法的建立及初步应用[J].中国畜牧兽医,2012,39(10)：45-49.
[81].范瑾,谭磊,仇旭升,等.传染性支气管炎病毒RT-LAMP检测方法的建立[J].中国兽医科学,2012,(11)：1163-1167.
[82]向智龙,卓建华,鲜思美,等.羊口疮病毒环介导等温扩增快速检测方法的建立及应用[J].中国兽医科学,2011,41(6)：588-592.
[83]杨春华,曹际娟,桂家祥,等.乳及乳制品中阪崎肠杆菌PCR-DHPLC检测新技术的建立[J].微生物学通报,2008,(11)：1845-1849.
[84]曹际娟,闫平平,徐君怡,等.单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立[J].生物技术通报,2008,S1：415-419.
[85]孙涛,徐彪,朱来华,等.禽肉产品中禽流感与新城疫多重PCR-DHPLC检测方法的建立[J].中国动物检疫,2011,(3)：41-45.
[86]Hussain K A, Burger D. In Vivo and in vitro characteristics of Contagious Ecthyma Virus isolations:host response mechanism[J].Vet Micro,1989,19:23-36.
[87]王志军.羊CE病的诊断和防治[J].河南畜牧兽医,1999,20(1)：23.
[88]Haig D M, Deane DL, Myatt N, et al. The activation status of ovine CD45R+and CD45R-efferent lymph T cells after orf virus reinfeetion[J]. J Comp Pathol,1996,115:163-174.
[89]Demartini J C, Pearson L D, Fiscus S A.Chromium-51-release assay of antibody and complement-mediated cytotoxicity for contagious ecthyma virus-infected cell[J]. Am J vet Res,1978,39:1922-1926.
[90]Azwai S M, Carter S D, Woldehiwet Z. Immune responses of the carnal to contagious ecthyma (orf) virus infection[J]. Vet Micro,1995,47:119-131.
[91]McKeever D J, Jenkinson D M, Hutchison G, et al. Studies of the pathogenesis of orf virus infectionin sheep[J]. J.Comp.Pathol.1988,99:317-328.
[92]Buddle B M, Pulford H D. Effect of passively avquired antibodies and vaccination on the immune response to contagious ecthyma virus[J]. Vet Microbiol,1984,9:515-522.
[93]Buddle B M, Deller R W, Schurig G G, et al. Heterogeneity of contagious ecma virus isolates[J]. Am J Vet Res,1984, 32(45):75-79.
[94]薛双虎,吕武夫,薛掌林,等.羊传染性脓疱皮炎病毒弱毒株的培育研究[J].甘肃畜牧兽医,1989,(3)：1-2.
[95]郭志宏,蒋元生.羊传染性脓疱病毒弱毒苗与其保护率的相关性[J].青海畜牧兽医杂志,1996,26(2)：28.
[96]詹述宣,王廷璞.羊CE基因工程苗的基础研究概述[J].西北民族学院学报,1999,20(12)：40-41.
[97]王延璞,吕武夫,薛双虎,等.羊CE弱毒苗对不同病毒株的交互免疫[J].甘肃畜牧兽医,1997,(6)：4-6.
[98]王延璞,薛双虎,吕武夫.羊CE皮炎的免疫特性[J].中国兽医科技,1990,(2)：15-17.
[99]Lederman E R, Green G M, DeGroot H E, et al. Progressive Orf virus infection in a patient with lymphoma:successful treatment using imiquimod[J]. Clinical Infection Diseases,2007,44:e100-e103.
[100]周永连,欧阳荣升,潘叔惠,等.速效碘杀灭羊传染性病毒试验[J].贵州畜牧兽医,1997,2：3.
[101]唐如勋.含醇利凡诺水溶液治疗羊CE病[J].中国兽医杂志,1996,22：40.
[102]王廷璞,董志辉.秃疮花对羊CE病毒抑制作用之测定[J].甘肃省畜牧兽医,1998,(1)：9-10.
[103]汪焕新.云南白药巧治羊CE[J].福建畜牧兽医,1998,(1)：33.
[104]张家确,吴景央.一起人兽共患羊传染性脓疱病的调查[J].中国兽医杂志,2006,42(9)：53-54.
[105]罗云,申之义.羊传染性脓疱病毒分离及生物学特性研究[J].中国动物检疫,2006,23(8)：25-27.
[106]Scagliarini A, Ciulli S, Batmani M. Characterisation Of immunodominant protein encoded By the F1L gene of orf virus strain isolated in Itatly[J]. Arch Virol,2002,147:1989-1995.
[107]A. Scagliarini, L. Gallina, F. Dal Pozzo, et al. Heparin binding activity of orf virus F1L protein[J]. Virus Research,2004, 105:107-112.
[108]Gallina L, Scagliarini A, Ciulli S, et al. Cloning and expression of the Orf virus F1L gene, Possible Use as a subunit vaccine[J]. Veterinary research communications,2004,28(1):291-293.
[109]邵洪泽,毛文智,宋阳,等.羊传染性脓疱病毒B2L基因片段重组腺病毒载体的构建[J].安徽农业科学,2010,38(13)：6722-6724.
[110]孔繁梅,史全军,宗晓龙,等.用牦牛睾丸细胞生产羊传染性脓疱皮炎疫苗效果观察[J].中国兽药杂志,2006,40(4)：58-59.
[111]邓小燕.抗寒基因表达载体构建及遗传转化研究[D].西南农业大学,2003.
[112]赵魁,贺文琦,高丰.羊传染性脓疱皮炎病毒研究进展[J].中国畜牧兽医,2008,35(11)：133-136.
[113]赵魁.羊传染性脓疱病毒重组DNA疫苗的构建与实验免疫研究[D].吉林大学博士论文,2010.
[114]王秋悦.柔嫩艾美耳球虫rhomboid基因重组卡介苗的构建及其免疫保护性研究[D].吉林大学博士论文,2009.
[115]尹春光,杜立新,赵桂平,等.Mx基因稀有密码子和mRNA结构及大肠杆菌表达优化[J].遗传,2009,(1)：75-82.
[116]王光祥,尚佑军,吕占禄,等.羊口疮病毒F1L蛋白二级结构分析与表位预测[J].中国人兽共患病学报,2012,(12)：1185-1190.
[117]吉艳红,尚佑军,王光祥,等.羊口疮病毒F1L基因的克隆及其B细胞表位预测[J].中国兽医科学,2010,(11)：1101-1105.
[118]李瑞芳,乔军,张辉,等.羊传染性脓疱病毒新疆流行株F1L基因克隆及表达[J].生物技术,2012,22(3)：13-16.
[119]Hosamani M, Scagliarini A, Bhanuprakash V, et al. Orf:an update on current research and future perspectives[J].Expert Rev Anti Infect Ther. 2009, (7):879-893.
[120]Gurnbrell RC, McGregor DA. Outbreak of severe fatal orf in lambs[J]. Vet. Rec,1997,141:150-151.
[121]Mazur C, Machado RD. Detection of contagious pustular dermatitis virus of goats in a severe outbreak[J]. Vet. Rec.1989, 125:419-420.
[122]郭素珍,张惠安,邝明慧,等.羊口疮病原的分离与鉴定研究[J].中国兽医科技,1987,17(4)：3,4.
[123]郭素珍,张惠安,黄守明,等.骆驼、绵羊、山羊口疮病原的血清学关系比较[J].中国兽医科技,1988,(5)：50-52.
[124]朱伟,熊毅,刘棋,等.快速鉴别诊断山羊痘病毒和羊口疮病毒二联PCR方法的建立[J].中国预防兽医学报,2007,(5)：394-396.
[125]李超,赵魁,赵权,等.羊传染性脓疱病毒SYBR Green I实时荧光定量PCR检测方法的建立[J].中国兽医科学,2010,40(3)：270-273.
[126]周支香,姜华,王际平,等.城LAMP技术检测单纯疱疹病毒-2型的初步应用[J].南华大学学报：医学版,2010,(2)：180-182.
[127]张如胜,宋克云,欧新华,等.志贺菌和肠侵袭性大肠埃希菌LAMP检测方法的建立及应用[J].国际检验医学杂志,2013,(1)：20-22.
[128]史亚东,董海聚,王荣军,等.微小隐孢子虫LAMP检测方法的探索[J].中国人兽共患病学报,2012,(12)：1195-1201.
[129]刘伟,侯丽萍,赵良娟,等.环介导等温扩增技术检测食品中铜绿假单胞菌[J].食品研究与开发,2012,(5)：140-142.
[130]陈金松黄丛林张秀海,等.环介导等温扩增技术检测含有CaMV35S的转基因玉米[J].华北农学报,2011,(4)：8-14.
[131]程荣华,邵洪泽,呼延含蓉,等.羊传染性脓疱病的诊断与防治[J].吉林农业,2010,(12)：203.
[132]王辉.羊传染性脓疱病毒F1L基因克隆、序列分析及核酸探针的研究[D].内蒙古农业大学硕士论文,2007.
[133]王光华,陆艳,王戈平,等.羊传染性脓疱诊断方法的建立[J].青海畜牧兽医杂志,2012,(5)：1-3.
[134]杨忠伟,冯秀丽,王忠,等.利用变性高效液相色谱方法进行HCM家系MYH7基因突变筛查研究[J].农垦医学,2012,(2)：127-129.
[135]白月,栾凤侠,高宏伟,等.应用多重PCR-DHPLC方法快速检测转基因马铃薯及EH92-527-1品系鉴定[J].中国马铃薯,2011,(3)：129-134.
[136]孙芳,赵玉玲,王子玲,等.DHPLC法和测序法检测结核分枝杆菌临床分离株链霉素的耐药性[J].医药论坛杂志,2011,(5)：15-19.
[137]孙涛,肖西志,徐彪,等.Ⅰ型鸭肝炎病毒PCR-DHPLC检测方法的建立[J].动物医学进展,2011,(7)：13-18.
[2]邵洪泽,刘云志,程荣华,等.羊传染性脓疱病毒的分离与鉴定[J].吉林畜牧兽医,2006,27(11)：11-12.
[3]赵魁,高丰,贺文琦,等.1株长春地区羊传染性脓疱病毒的分离鉴定及F1L基因的克隆与序列分析[J].中国兽医学报,2010,(7)：931-935.
[4]殷震,刘景华.动物病毒学[M].第二版,北京：科学出版社,1997,977-980.
[5]王光华,陆艳,王戈平,等.羊传染性脓疱诊断方法的建立[J].青海畜牧兽医杂志,2012,05：1-3.
[6]陈光辉.一起羊传染性脓疱的防治[J].甘肃畜牧兽医,2004,(4)：32-33.
[7]周江名,夏刚位.羊传染性脓疱综合防治[J].四川畜牧兽医,2011,(6)：54-55.
[8]闫喜军,卢金明,王艳华.羊传染性脓疱病的治疗[J].养殖技术顾问,2004,(2)：29.
[9]李瑞芳,乔军,张辉,等.羊传染性脓疱病毒新疆流行株F1L基因克隆及表达[J].生物技术,2012,(3)：17-20.
[10]王辉,赵晓军,易建刚,等.羊传染性脓疱病毒内蒙分离株F1L基因的克隆与序列分析[J].中国动物检疫,2007,(6)：26-27.
[11]赖自武,郑建珍,陈家俊.羊传染性脓疱的综合防治[J].云南农业,2005,(5)：14.
[12]张克山,何继军,尚佑军,等.羊传染性脓疱病毒湖北株的鉴定及分子特征分析[J].畜牧兽医学报,2010,(9)：1154-1157.
[13]于恩庶,林继煌,陈观今.中国人兽共患病学(第二版)[M].福州：福建科学技术出版社,1996,753-758.
[14]中国农业科学院哈尔滨兽医研究所.动物传染病学[M].北京：中国农业出版社,1998,363-365.
[15]蔡宝祥.家畜传染病学[M].北京：中国农业出版社,2001,157-159.
[16]蔡宝祥,殷震,谢三星,等.动物传染病诊断学[M].江苏科学技术出版社,1993,187-188.
[17]Straten M van, Friedgut O. Outbreak of contagious ecthyma in carnal in srael[J]. Vet Reco,2001,148:150-151.
[18]Kummeneje K, Krogsrud J. Contagious ecthyma (Orf) in the musk ox [J]. Acta Vet Scand,1978,19:461-462.
[19]Kummeneje K, Krogsrud J. Contagious ecthyma (Orf) in reindeer[J]. Vet Rec,1979,105:60-61.
[20]WilkinsonG T, Prydie J, Scarnell J. Possible"orf'(contagious pustular dermatitis, contagious ecthyma of sheep) infection in the dog[J]. Vet Rec,1970,87:766-767.
[21]Nollens H H, Jacobson E R, Gulland F M D, et al. Pathology and preliminary characterization of a parapoxvirus isolated from a california sea lion (Zalophus californianus)[J]. Journal of Wildlife Diseases,2006,42(1):23-32.
[22]Becher P, Konig M, Muller G, et al. Characterization of sealpoxvirus, a separate member of the parapoxvirus [J].Arch Virol, 2002,147:1133-1140.
[23]王积禄,等.羊传染性口膜炎[J].中国畜牧兽医,1962,(2)：10-11.
[24]陈燕军.羊口疮-病原学及试验病理学研究[J].兽医科技杂志,1982,(6)：2-8.
[25]Groves R W, Wilson-Jones E, MacDonald D M. Human orf and milkers'nodule:a clinicopathologic study[J]. J Amer Acad Dermatol,1991,25:706-711.
[26]Kuhl J T, Huerter C J, Hashish H. A case of human orf contracted from a deer[J]. Cutis.2003,71(4):288-289.
[27]甘肃农业大学主编.家畜传染病学[M].北京：农业出版社,1984,365-367.
[28]Czerny C P, Waldmann R, Scheubeck T. Identification of three distinct antigenic sites in parapoxvirus[J]. Arch Virol,1997, 142:807-821.
[29]A Boxton, et al. Animal microbiology[M].1977,670-691.
[30]Rossi G A. A daptaation of the virus of contagious ecthyma to cellular substrates of avian origin[J]. Vet Ital,1973,24: 218-222.
[31]Robinson A J,Barns G,Fraaer K,et.al. Conservation and Variation in orf virus genores[J]. Virology,1987,157:13-23.
[32]Greig A S. Contagious ecthyma of sheep Ⅱ.In vitro cultivation of the virus[J]. Can J comp Med,1957,21:304-308.
[33]赵立志,薛双虎,吕武夫,等.羊传染性农疱皮炎病毒的细胞培养[J].中国兽医杂志,1991,17(8)：12-13.
[34]何水林,陈杰,蔡玉书.羊口疮病毒的分离鉴定[J].中国兽医科技,2002,9：22-23.
[35]Pye D. Vaccination of sheep with cell culture grown orf virus[J]. Aust Vet J,1990,67:182-186.
[36]Straten M van,Friedgut O. Outbreak of contagious ecthyma in camal in Israel [J]. Vet Reco,2001,148:150-151.
[37]Plowright W, Whitcomb M A, Ferris R D. Studies with a strain of contagious pustular dermatitis virus in tissue culture[J]. Arch Ges virus forsch,1959,9:214-231.
[38]白吉荣,沈正达,王锡祯,等.羊口疮病毒在单层细胞上繁殖特性的研究[J].甘肃农业大学学报,1985,(4)：109-116.
[39]庞理化,程庆华,侯秀芬,等.羊口疮病毒的组织培养初报[J].中国兽医杂志,1982,(3)：66-69.
[40]詹述宣,薛双虎,薛掌林.犊牛睾丸细胞培养羊接触传染性脓疱皮炎病毒的研究[J].兽医科技杂志,1983,(7)：5-7.
[41]Robinson A J, Ellis G, Balassu T C. The genome of orf virus:restrictionendonuclease analysis of viral DNA isolated from lesions of orf in sheep[J]. Arch. Virol,1982,71,43-55.
[42]Gassmann U, Wyler R, Wittek R. Analysis of parapoxvirus genomes[J]. Arch. Virol.,1985,83:17-31.
[43]Cottone R, Biittner M, Bauer B, et al. Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[J]. Virus Res,1998,56:53-67.
[44]Mercer A A, Fraser K, Barns G, et al. The structure and cloning of orf virus DNA[J]. Virology,1987,157:1-12.
[45]Haig D M, Mcinnes C J, Thomson J, et al. The orf virus OV20.OL gene product is involved in interferon resistance and inhibit san interferon 2 inducible, double 2st randed RNA2 dependent kinase[J]. Immunology,1998,93:335-340.
[46]Spehner D, De Carlo S, Drillien R, et al. Appearance of the bona fide spiral tubule of Orf virus is dependent on an ntact 10-kilodalton viral protein[J]. Virology,2004,8085-8093.
[47]Cottone R, But tner M, Mclnnes C J, et al. Orf virus encodes afunctional dU TPase gene[J].General Virology,2002,83: 1043-1048.
[48]Marsland B J, Tisdall D J, Heat h D D, et al. Const ruction of a recombinant orf virus t hat expresses an Echinococcus granulosus vaccine antigen from a novel genomic insertion site[J]. Arch Virol,2003,148 (3):555-562.
[49]Einar G, Torfason, Sigrun. Polymerase chain reaction for laboratory diagnosis of orf virus infection[J]. J Clinic Virol,2002, 24:79-84.
[50]Sullivan J T, Mercer A A, Fleming S B, et al. Identification and characterization of an orf virus homologue of vaccinia virus gene encoding t he major envelop antigen p37k[J].Virology,1994,202:968-973.
[51]Becher P, Konig M, Muller G, et al. Characterization of sealpox virus, a separate member of the parapoxvirus [J]. Arch Vir ol,2002,147:1133-1140.
[52]Wise LM, Veikkola T, Mercer AA, et al. Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1[J]. Proc Natl Acad Sci USA,1999,96:3071-3076.
[53]Seet BT, McCaughan CA, Handel TM, et al. Analysis of an orf virus chemokine-binding protein:Shifting ligand specificiti es among a family of poxvirus viroceptors[J]. PNAS,2003,100 (25):15137-15142.
[54]Haig DM, Mclnnes CJ. Immunity and counter-immunity during infection with the parapoxvirus orf virus[J]. Virus Res,200 2a,88:3-16.
[55]王廷璞,赵菲佚,安建平,等.羊传染性脓疱病毒42K囊膜蛋白基因克隆及表达[J].中国预防兽医学报,2006,28(1)：29-32.
[56]陈燕军.羊口疮的研究—病原学及试验病理学研究[J].兽医科技杂志,1982,6：2-8.
[57]白积荣,沈正达,王锡贞,等.羊口疮病毒形态发生的电镜观察[J].中国兽医科技,1985,(11)：34-36.
[58]Harkness J W, Scott A C, Hebert C N. Electron microscopy in the rapid diagnosis of ORF[J]. British Vet J,1977,133: 81-87.
[59]陈燕军.羊口疮的研究—血清学方法的研究[J].兽医科技杂志,1982,7：14-19.
[60]薛掌林,薛双虎,吕武夫.中和试验对羊传染性脓疱皮炎抗体的检测[J].甘肃畜牧兽医,1990,2：13-14.
[61]Robinson A J, Ellis G,Balassu T C.The genome of orfvirus:Restrictionendonucle seanalys is of viral DNA isolatedfrom lesion so for finsheep[J]. ArchivesofVirology,1982,71:43-55.
[62]王延璞,薛双虎,吕武夫.酶联免疫吸附试验检测CEV[J].甘肃畜牧兽医,1995,25(1)：3-4.
[63]Chin JC, Petersen RK. Comparison of native and subunit antigens as ELISA reagents for thedetection of antibodies against scabby mouth virus[J]. Vet Microbiol,1995,46:327-334.
[64]Chand P, Kitching R P, Black D N. Western blot analysis of virus-specific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep[J]. Epidemiology & Infection,1994,113:377-385.
[65]伍新尧,罗超,等.分子遗传学与基因工程[M].郑州：河南医科大学出版社,1997,157-170.
[66]G H凯勒,M M马纳克.DNA探针技术[M].北京：科学出版社,1992,186-187.
[67]Robinson A J, Ballassu T C,Contagious Pustular dermatitis (Orf) [J]. Vet Bull,1981,51:771-782.
[68]Andrew A Mencr, Kate Fraser, GillianBarns, et al. the structure and cloning of orf viring DNA[J]. virology,1987,157: 11-21.
[69]王廷璞,赵菲佚,孙春香,等.光敏生物素和DIG标记Orf病毒DNA探针的研究[J].生物技术,2002,12(6)：21-22.
[70]李会荣,龚振华,郑增忍.应用PCR检测羊接触CE性皮炎病毒[J].中国兽医科技,2003,33：10-13.
[71]刘云志,邵洪泽,程荣华,等.羊传染性脓疱病毒PCR检测方法的建立及应用[J].吉林畜牧兽医,2007,28(3)：7-9.
[72]Einar G.Torfason, sigrun Gu nadottir.Polymerase chain reaction for laboratory diagnosis of orf virus infections[J]. J chin virol,2002,24:79-84.
[73]Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res. 2000,28:63.
[74]Kurosaki Y, Takada A, Ebihara H, et al. Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification[J]. Journal of virological methods.2007,141:78-83.
[75]Yoshino M, Watari H, Kojima T, et al. Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification[J]. Microbiology and immunology,2009,53:375-383.
[76]Kono T, Savan R, Sakai M, et al. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification[J]. Journal of virological methods,2004,115:59-65.
[77]Aryan E, Makvandi M, Farajzadeh A, et al. A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex[J]. Microbiological Research,2010,165:211-220.
[78]Horibe D, Ochiai T, Shimada H, et al. Rapid detection of metastasis of gastric cancer using reverse transcription loop-mediated isothermal amplification[J]. Int J Cancer.2007 Mar 1,120(5):1063-1069.
[79]Saleh M, Soliman H and El-Matboulicrossref M. Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish[J]. BMC Veterinary Research,2008,4:31.
[80]胡嘉欣,温永俊,霍志云,等.犬瘟热病毒RT-LAMP检测方法的建立及初步应用[J].中国畜牧兽医,2012,39(10)：45-49.
[81].范瑾,谭磊,仇旭升,等.传染性支气管炎病毒RT-LAMP检测方法的建立[J].中国兽医科学,2012,(11)：1163-1167.
[82]向智龙,卓建华,鲜思美,等.羊口疮病毒环介导等温扩增快速检测方法的建立及应用[J].中国兽医科学,2011,41(6)：588-592.
[83]杨春华,曹际娟,桂家祥,等.乳及乳制品中阪崎肠杆菌PCR-DHPLC检测新技术的建立[J].微生物学通报,2008,(11)：1845-1849.
[84]曹际娟,闫平平,徐君怡,等.单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立[J].生物技术通报,2008,S1：415-419.
[85]孙涛,徐彪,朱来华,等.禽肉产品中禽流感与新城疫多重PCR-DHPLC检测方法的建立[J].中国动物检疫,2011,(3)：41-45.
[86]Hussain K A, Burger D. In Vivo and in vitro characteristics of Contagious Ecthyma Virus isolations:host response mechanism[J].Vet Micro,1989,19:23-36.
[87]王志军.羊CE病的诊断和防治[J].河南畜牧兽医,1999,20(1)：23.
[88]Haig D M, Deane DL, Myatt N, et al. The activation status of ovine CD45R+and CD45R-efferent lymph T cells after orf virus reinfeetion[J]. J Comp Pathol,1996,115:163-174.
[89]Demartini J C, Pearson L D, Fiscus S A.Chromium-51-release assay of antibody and complement-mediated cytotoxicity for contagious ecthyma virus-infected cell[J]. Am J vet Res,1978,39:1922-1926.
[90]Azwai S M, Carter S D, Woldehiwet Z. Immune responses of the carnal to contagious ecthyma (orf) virus infection[J]. Vet Micro,1995,47:119-131.
[91]McKeever D J, Jenkinson D M, Hutchison G, et al. Studies of the pathogenesis of orf virus infectionin sheep[J]. J.Comp.Pathol.1988,99:317-328.
[92]Buddle B M, Pulford H D. Effect of passively avquired antibodies and vaccination on the immune response to contagious ecthyma virus[J]. Vet Microbiol,1984,9:515-522.
[93]Buddle B M, Deller R W, Schurig G G, et al. Heterogeneity of contagious ecma virus isolates[J]. Am J Vet Res,1984, 32(45):75-79.
[94]薛双虎,吕武夫,薛掌林,等.羊传染性脓疱皮炎病毒弱毒株的培育研究[J].甘肃畜牧兽医,1989,(3)：1-2.
[95]郭志宏,蒋元生.羊传染性脓疱病毒弱毒苗与其保护率的相关性[J].青海畜牧兽医杂志,1996,26(2)：28.
[96]詹述宣,王廷璞.羊CE基因工程苗的基础研究概述[J].西北民族学院学报,1999,20(12)：40-41.
[97]王延璞,吕武夫,薛双虎,等.羊CE弱毒苗对不同病毒株的交互免疫[J].甘肃畜牧兽医,1997,(6)：4-6.
[98]王延璞,薛双虎,吕武夫.羊CE皮炎的免疫特性[J].中国兽医科技,1990,(2)：15-17.
[99]Lederman E R, Green G M, DeGroot H E, et al. Progressive Orf virus infection in a patient with lymphoma:successful treatment using imiquimod[J]. Clinical Infection Diseases,2007,44:e100-e103.
[100]周永连,欧阳荣升,潘叔惠,等.速效碘杀灭羊传染性病毒试验[J].贵州畜牧兽医,1997,2：3.
[101]唐如勋.含醇利凡诺水溶液治疗羊CE病[J].中国兽医杂志,1996,22：40.
[102]王廷璞,董志辉.秃疮花对羊CE病毒抑制作用之测定[J].甘肃省畜牧兽医,1998,(1)：9-10.
[103]汪焕新.云南白药巧治羊CE[J].福建畜牧兽医,1998,(1)：33.
[104]张家确,吴景央.一起人兽共患羊传染性脓疱病的调查[J].中国兽医杂志,2006,42(9)：53-54.
[105]罗云,申之义.羊传染性脓疱病毒分离及生物学特性研究[J].中国动物检疫,2006,23(8)：25-27.
[106]Scagliarini A, Ciulli S, Batmani M. Characterisation Of immunodominant protein encoded By the F1L gene of orf virus strain isolated in Itatly[J]. Arch Virol,2002,147:1989-1995.
[107]A. Scagliarini, L. Gallina, F. Dal Pozzo, et al. Heparin binding activity of orf virus F1L protein[J]. Virus Research,2004, 105:107-112.
[108]Gallina L, Scagliarini A, Ciulli S, et al. Cloning and expression of the Orf virus F1L gene, Possible Use as a subunit vaccine[J]. Veterinary research communications,2004,28(1):291-293.
[109]邵洪泽,毛文智,宋阳,等.羊传染性脓疱病毒B2L基因片段重组腺病毒载体的构建[J].安徽农业科学,2010,38(13)：6722-6724.
[110]孔繁梅,史全军,宗晓龙,等.用牦牛睾丸细胞生产羊传染性脓疱皮炎疫苗效果观察[J].中国兽药杂志,2006,40(4)：58-59.
[111]邓小燕.抗寒基因表达载体构建及遗传转化研究[D].西南农业大学,2003.
[112]赵魁,贺文琦,高丰.羊传染性脓疱皮炎病毒研究进展[J].中国畜牧兽医,2008,35(11)：133-136.
[113]赵魁.羊传染性脓疱病毒重组DNA疫苗的构建与实验免疫研究[D].吉林大学博士论文,2010.
[114]王秋悦.柔嫩艾美耳球虫rhomboid基因重组卡介苗的构建及其免疫保护性研究[D].吉林大学博士论文,2009.
[115]尹春光,杜立新,赵桂平,等.Mx基因稀有密码子和mRNA结构及大肠杆菌表达优化[J].遗传,2009,(1)：75-82.
[116]王光祥,尚佑军,吕占禄,等.羊口疮病毒F1L蛋白二级结构分析与表位预测[J].中国人兽共患病学报,2012,(12)：1185-1190.
[117]吉艳红,尚佑军,王光祥,等.羊口疮病毒F1L基因的克隆及其B细胞表位预测[J].中国兽医科学,2010,(11)：1101-1105.
[118]李瑞芳,乔军,张辉,等.羊传染性脓疱病毒新疆流行株F1L基因克隆及表达[J].生物技术,2012,22(3)：13-16.
[119]Hosamani M, Scagliarini A, Bhanuprakash V, et al. Orf:an update on current research and future perspectives[J].Expert Rev Anti Infect Ther. 2009, (7):879-893.
[120]Gurnbrell RC, McGregor DA. Outbreak of severe fatal orf in lambs[J]. Vet. Rec,1997,141:150-151.
[121]Mazur C, Machado RD. Detection of contagious pustular dermatitis virus of goats in a severe outbreak[J]. Vet. Rec.1989, 125:419-420.
[122]郭素珍,张惠安,邝明慧,等.羊口疮病原的分离与鉴定研究[J].中国兽医科技,1987,17(4)：3,4.
[123]郭素珍,张惠安,黄守明,等.骆驼、绵羊、山羊口疮病原的血清学关系比较[J].中国兽医科技,1988,(5)：50-52.
[124]朱伟,熊毅,刘棋,等.快速鉴别诊断山羊痘病毒和羊口疮病毒二联PCR方法的建立[J].中国预防兽医学报,2007,(5)：394-396.
[125]李超,赵魁,赵权,等.羊传染性脓疱病毒SYBR Green I实时荧光定量PCR检测方法的建立[J].中国兽医科学,2010,40(3)：270-273.
[126]周支香,姜华,王际平,等.城LAMP技术检测单纯疱疹病毒-2型的初步应用[J].南华大学学报：医学版,2010,(2)：180-182.
[127]张如胜,宋克云,欧新华,等.志贺菌和肠侵袭性大肠埃希菌LAMP检测方法的建立及应用[J].国际检验医学杂志,2013,(1)：20-22.
[128]史亚东,董海聚,王荣军,等.微小隐孢子虫LAMP检测方法的探索[J].中国人兽共患病学报,2012,(12)：1195-1201.
[129]刘伟,侯丽萍,赵良娟,等.环介导等温扩增技术检测食品中铜绿假单胞菌[J].食品研究与开发,2012,(5)：140-142.
[130]陈金松黄丛林张秀海,等.环介导等温扩增技术检测含有CaMV35S的转基因玉米[J].华北农学报,2011,(4)：8-14.
[131]程荣华,邵洪泽,呼延含蓉,等.羊传染性脓疱病的诊断与防治[J].吉林农业,2010,(12)：203.
[132]王辉.羊传染性脓疱病毒F1L基因克隆、序列分析及核酸探针的研究[D].内蒙古农业大学硕士论文,2007.
[133]王光华,陆艳,王戈平,等.羊传染性脓疱诊断方法的建立[J].青海畜牧兽医杂志,2012,(5)：1-3.
[134]杨忠伟,冯秀丽,王忠,等.利用变性高效液相色谱方法进行HCM家系MYH7基因突变筛查研究[J].农垦医学,2012,(2)：127-129.
[135]白月,栾凤侠,高宏伟,等.应用多重PCR-DHPLC方法快速检测转基因马铃薯及EH92-527-1品系鉴定[J].中国马铃薯,2011,(3)：129-134.
[136]孙芳,赵玉玲,王子玲,等.DHPLC法和测序法检测结核分枝杆菌临床分离株链霉素的耐药性[J].医药论坛杂志,2011,(5)：15-19.
[137]孙涛,肖西志,徐彪,等.Ⅰ型鸭肝炎病毒PCR-DHPLC检测方法的建立[J].动物医学进展,2011,(7)：13-18.