CN106442968B - 猪繁殖与呼吸综合征血清学鉴别诊断试剂盒 - Google Patents
猪繁殖与呼吸综合征血清学鉴别诊断试剂盒 Download PDFInfo
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Abstract
本发明介绍一种猪繁殖与呼吸综合征病毒（PRRSV）血清学鉴别诊断试剂盒。其中，利用多肽1为包被抗原建立的间接ELISA方法，可同时检测美洲型和欧洲型PRRSV血清学抗体；利用多肽2为包被抗原建立的间接ELISA方法，仅针对美洲型PRRSV抗体；利用多肽3为包被抗原建立的间接ELISA方法，仅针对欧洲型PRRSV抗体；利用多肽4为包被抗原建立的间接ELISA方法，仅针对高致病PRRSV抗体。利用本发明建立的猪繁殖与呼吸综合征血清学鉴别诊断试剂盒可同时对当前我国猪群中的PRRSV血清学抗体进行评价。本方法简便快捷，可有效用于PRRSV的临床血清学诊断。
Description
技术领域
本发明属于动物传染病学领域，具体涉及一种不同类型猪繁殖与呼吸综合征病毒（PRRSV）血清学鉴别诊断试剂盒。
背景技术
猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒（porcine reproductive andrespiratory syndrome virus， PRRSV）引起的，可导致妊娠母猪发热、厌食及繁殖障碍（如流产、早产、死胎、木乃伊胎），新生及断奶仔猪高死亡率的仔猪呼吸道疾病，俗称猪“蓝耳病”。根据血清学和基因组特征分析，可将PPRSV进一步分为欧洲型（代表株为LV株）和美洲型（代表株为VR2332株）。2006年，我国南方地区出现以高热、高发病率和高死亡率为特征的传染病，经农业部最终确定“高热病”为“高致病性蓝耳病”，即HP-PRRSV（属于美洲型PRRSV）。对HP-PRRSV进行基因组分析比较发现，HP-PRRSV在非结构蛋白2（Nsp2）存在连续核苷酸（约87bp，29个氨基酸）缺失。2008年，欧洲型PRRSV在我国福建和浙江猪群中首次发现报道，随后在我国多省市发现欧洲型PRRSV感染阳性的报道。
猪繁殖与呼吸综合征病毒属于网巢病毒目（Nidovirales）动脉炎病毒科（Arteriviridae）动脉炎病毒属（Arterivirus），该类病毒为单股正链RNA病毒。PRRSV基因组长约15kb，分别为（5’端至3’端）：非结构蛋白ORF1（ORF1a和ORF1b）、结构蛋白ORF2a（GP2a）、ORF2b（GP2b）、ORF3（GP3）、ORF4（GP4）、ORF5（GP5）、ORF6（M）和ORF7（N）。PRRSV基因组结构和其他动脉炎病毒组基因组结构相似，在病毒基因组的5’端有帽子结构，3’端多聚腺苷酸化。其非结构蛋白ORF1占基因组全长的75%左右，包含有PRRSV复制所需的酶类，参与病毒的增殖过程，可进一步细分为Nsp1、Nsp2、Nsp3、Nsp4、Nsp5、Nsp6、Nsp7、Nsp9、Nsp1和Nsp11共10种非结构蛋白。结构蛋白中ORF2-ORF6编码病毒的囊膜蛋白，其中ORF2-ORF5编码的蛋白均为糖基化蛋白（GP2、GP3、GP4和GP5）而ORF6编码的M蛋白为非糖基化蛋白；ORF7编码病毒的核衣壳蛋白（N），有较强的免疫原性。
目前，我国猪群中PPRSV感染的类型较为复杂，为了更好的预防和控制PRRSV的流行，需要对猪群PRRSV的进行科学有效区分。从病原学来看，根据欧洲型和美洲型PRRSV的基因组差异已见有RT-PCR和实时荧光定量PCR方法。此外，关于HP-PRRSV和美洲型PRRSV的基因组差异（主要是Nsp2）的鉴别诊断方法已有RT-PCR和实时荧光定量PCR方法，但是上述实验方法对实验操作者和实验仪器设备要求较高，不适宜与临床现场使用。从血清学来看，已见有欧洲型和美洲型PRRSV鉴别诊断ELISA方法报道；此外，也见有HP-PRRSV和美洲型PRRSV的基因组差异鉴别诊断ELISA报道。但是，当前未见可同时区分欧洲型PRRSV、美洲型PRRSV和HP-PRRSV的血清学鉴别诊断试剂盒报道。
抗原表位（antigen epitope）是指抗原分子表面具有刺激机体产生抗体或致敏淋巴细胞并能被其识别的免疫活性区，又称抗原决定簇（antigenic determination）。根据表位与受体细胞结合部位的差异，表位可分为B细胞抗原表位和T细胞抗原表位；根据表位结构不同可分为连续抗原表位（又称线性表位，由肽链上连续氨基酸组成，主要是T细胞表位和部分B细胞表位）和不连续抗原表位（又称构象表位，由空间上相近但顺序上不连续的氨基酸组成，仅见B细胞表位）。本研究根据报道的PRRSV表位进行合成多肽，分别建立美洲型和欧洲型PRRSV都可检测的间接ELISA方法（针对N蛋白）；建立针对美洲型PRRSV型保守表位的间接ELISA方法（针对GP5蛋白）；建立针对欧洲型PRRSV型保守表位的间接ELISA方法（针对ORF4蛋白）；针对HP-PRRSV的Nsp2连续核苷酸缺失区编码的氨基酸序列的间接ELISA方法。经组合后建立可针对PRRSV当前流行的不同类型（美洲型、欧洲型、HP-PRRSV）的血清学抗体进行科学有效区分的ELISA试剂盒。本发明可填补相关研究空白，为我国科学防控PRRSV提供新的参考。
发明内容
本发明的目的在于提供一种能够区分欧洲型PRRSV、美洲型PRRSV和HP-PRRSV血清学抗体的ELISA试剂盒，为大规模现场临床猪血清样品的检测以及开展PRRSV流行病学调查提供一种不同类型PRRSV鉴别诊断血清学ELISA试剂盒。
为实现上述目的，本发明采用如下技术方案：
猪繁殖与呼吸综合征血清学鉴别诊断试剂盒，所述试剂盒中含有4条抗原多肽，其序列为：多肽1：PEKPHFPLATEDDVRHH；多肽2 ：LRPHGVSAAQEEIPFGLSSQ；多肽3：EGHLIDLKRV；多肽4：RPMTPLSEPIFLSAPRHKFQQVEEANPAT。
以合成多肽为包被抗原的间接ELISA方法的操作程序：
（1）包被：将合成多肽用包被液稀释成一定浓度，包被酶标板，100μL/孔，4℃过夜，次日弃去包被液，用PBST洗涤3次，每次5min，250μL/孔，最后一次拍干。
（2）封闭：每孔加入200μL封闭液，37℃封闭2h（或4℃封闭过夜），再弃去封闭液，用PBST洗涤3次，每次5min，250μL/孔，最后一次拍干。
（3）加血清：血清用抗体稀释液按一定倍数稀释，加入酶标板中，100μL/孔，同时设阴性对照孔，阳性对照孔和空白对照孔，37℃孵育2h，弃去液体，用PBST洗涤3次，每次5min，250μL/孔，最后一次拍干。
（4）加酶标二抗：将酶标二抗用二抗稀释液（也可用PBST）按一定倍数稀释（商品化试剂可直接按照推荐的浓度稀释），加入酶标板中，100μL/孔，37℃孵育2h，弃去液体，用PBST洗涤3次，每次5min，250μL/孔，最后一次拍干。
（5）TMB反应液显色：将TMB反应液加入酶标板上，100μL/孔，室温避光反应15min。
（6）加入终止液：TMB显色后加入终止液，2M硫酸，100μL/孔。
（7）酶标仪测定：用酶标仪测定OD450值。
本发明中将包被时间明确为4℃包被过夜；封闭时间明确为4℃封闭过夜；一抗孵育时间明确为37℃孵育2h；二抗孵育时间明确为37℃孵育90min；显色7 min后测定OD450值。
本发明为了统一，在对30份阴性血清进行检测时，对30份进行检测后计算得到OD450nm值的平均数（X），标准方差（SD）。本发明统一规定，临界值（C）=平均数（X）+3标准方差（SD）。即本实验建立的ELISA对临床样品进行检测OD450nm值≧临界值（C）判为阳性，其他均判为阴性。
本发明的优点在于：
利用本发明建立的猪繁殖与呼吸综合征血清学鉴别诊断试剂盒可针对当前我国流行的PRRSV的不同类型（美洲型、欧洲型和HP-PRRSV）进行有效区分，开展PRRSV流行病学调查提供新的检测手段，可用于一线临床现场快速检测PRRSV的感染类型，为指导临床PRRSV疫苗的科学使用提供有益参考。试剂盒中仅仅采用了4条抗原多肽，就可以完成对不同类型（美洲型、欧洲型、HP-PRRSV）的血清学抗体的区分。本发明使用的多肽特异性强，来源稳定（易于专业公司合成获得），建立的ELISA方法较常规蛋白表达纯化后建立的蛋白方便快捷，鉴别试剂盒开发过程中重复性和稳定性更强，便于商品化推广使用。
附图说明
图1 欧洲型PRRSV和美洲型PRRSV的N蛋白氨基酸分析，图中“1NP-”表示的是欧洲型PRRSV；“2NP-”表示的是美洲型PRRSV。
图2 欧洲型PRRSV的ORF4抗原表位分析。
图3 美洲型PRRSV的GP5抗原表位分析。
图4经典型PRRSV和HP-PRRSV的非结构蛋白Nsp2差异区。
具体实施方式
实施例1
1试剂
ELISA平板购自Costar；HRP标记的山羊抗猪酶标二抗购自Abcam；ELISA显色液TMB购自武汉博士德生物工程有限公司。碳酸盐包被液、抗体稀释液和封闭液均购自碧云天生物技术研究所。
2血清
欧洲型PRRSV、美洲型PRRSV、HP-PRRSV、猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清由福建省农业科学院畜牧兽医研究所制备并保存。
3 合成多肽为抗原建立的间接ELISA方法
3．1 检测欧洲型PRRSV和美洲型PRRSV的多肽1建立的间接ELISA：
通过对欧洲型PRRSV和美洲型PRRSV基因组的ORF7编码区（N蛋白）核苷酸序列的抗原表位进行分析研究发现，其存在一个50PEKPHFPLATEDDVRHH66，该表位在欧洲型PRRSV和美洲型PRRSV编码的ORF7基因上较为保守。
3.1.1 多肽1的合成
对该表位多肽1经生工生物工程(上海)股份有限公司合成后，合成多肽的纯度＞95%。对合成的多肽溶解成母液（1mg）备用。
3.1.2 ELISA条件的确定
将抗原母液按照终浓度（1000ng、500ng、250ng、125 ng、62.5 ng和31.25 ng），将一抗（猪血清）按照（1:10、1:20、1:40、1:80、1:160和1:320）进行稀释，HRP标记的山羊抗猪酶标二抗按照说明书推荐的1:5000稀释。优化后的ELISA条件为，包被多肽1 250ng、血清稀释度为1：80、酶标二抗浓度1：5000即可获得最大化P/N值。对30份进行检测后计算得到OD450nm值的平均数（X）=0.2309、标准方差（SD）=0.0314，得到本研究的临界值（C）=0.3251。即本实验建立的ELISA对临床样品进行检测OD450nm值≧0.3251判为阳性，其他均判为阴性。
3．1.3 特异性实验
利用优化后的条件，对欧洲型PRRSV、美洲型PRRSV、HP-PRRSV、猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清进行检测。结果，欧洲型PRRSV、美洲型PRRSV、HP-PRRSV阳性血清OD450nm值分别为0.801、0.986和1.018；猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清均小于0.3251（结果见表1-1）。表明本研究建立的方法特异性强。
表1-1 特异性实验
3．1.4 重复性实验
利用优化后的条件建立的ELISA条件进行检测，获得组间变异系数在1.16%～4.93%之间，表明建立的ELISA具有良好的重复性。
3．1.5 临床样品检测
利用建立的ELISA方法对本收集的35份猪血清进行检测，其中阳性32份，阳性率为91.42%（32/35）。
4．1 检测欧洲型PRRSV多肽2建立的间接ELISA：
通过对欧洲型PRRSV 的ORF4编码区蛋白的抗原表位进行分析研究发现，其存在一个53 LRPHGVSAAQEEIPFGLSSQ 72，该表位在欧洲型PRRSV较为保守，和美洲型PRRSV差异极大。
4.1.1 多肽2的合成
对该表位多肽2经生工生物工程(上海)股份有限公司合成后，合成多肽的纯度＞95%。对合成的多肽溶解成母液（1mg）备用。
4.1.2 ELISA条件的确定
将抗原母液按照终浓度（1000ng、500ng、250ng、125 ng、62.5 ng和31.25 ng），将一抗（猪血清）按照（1:10、1:20、1:40、1:80、1:160和1:320）进行稀释，HRP标记的山羊抗猪酶标二抗按照说明书推荐的1:5000稀释。优化后的ELISA条件为，包被多肽2 500ng、血清稀释度为1：160、酶标二抗浓度1：5000即可获得最大化P/N值。对30份进行检测后计算得到OD450nm值的平均数（X）=0.2124、标准方差（SD）=0.0289，得到本研究的临界值（C）=0.2991。即本实验建立的ELISA对临床样品进行检测OD450nm值≧0.2991判为阳性，其他均判为阴性。
4．1.3 特异性实验
利用优化后的条件，对欧洲型PRRSV、美洲型PRRSV、HP-PRRSV、猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清进行检测。结果，欧洲型PRRSV、美洲型PRRSV、HP-PRRSV阳性血清OD450nm值分别为0.827、0.253和0.256；猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清均小于0.2991（结果见表1-2）。表明本研究建立的方法特异性强。
表1-2 特异性实验
4．1.4 重复性实验
利用优化后的条件建立的ELISA条件进行检测，获得组间变异系数在1.02%～4.32%之间，表明建立的ELISA具有良好的重复性。
4．1.5 临床样品检测
利用建立的ELISA方法对本收集的35份猪血清进行检测，其中欧洲型PRRSV阳性2份，阳性率为5.71%（2/35）；且这2份阳性血清在建立的多肽1为抗原包被的间接ELISA中检测也为阳性，阳性符合率为100%。
5．1 检测美洲型PRRSV多肽3建立的间接ELISA：
通过对美洲型PRRSV 的GP5编码区蛋白的抗原表位进行分析研究发现，其存在一个169EGHLIDLKRV178，该表位在美洲型PRRSV较为保守，和欧洲型PRRSV差异极大。
5.1.1 多肽3的合成
对该表位多肽3经生工生物工程(上海)股份有限公司合成后，合成多肽的纯度＞95%。对合成的多肽溶解成母液（1mg）备用。
5.1.2 ELISA条件的确定
将抗原母液按照终浓度（1000ng、500ng、250ng、125 ng、62.5 ng和31.25 ng），将一抗（猪血清）按照（1:10、1:20、1:40、1:80、1:160和1:320）进行稀释，HRP标记的山羊抗猪酶标二抗按照说明书推荐的1:5000稀释。优化后的ELISA条件为，包被多肽3 500ng、血清稀释度为1：160、酶标二抗浓度1：5000即可获得最大化P/N值。对30份进行检测后计算得到OD450nm值的平均数（X）=0.2657、标准方差（SD）=0.0334，得到本研究的临界值（C）=0.3659。即本实验建立的ELISA对临床样品进行检测OD450nm值≧0.3659判为阳性，其他均判为阴性。
5．1.3 特异性实验
利用优化后的条件，对欧洲型PRRSV、美洲型PRRSV、HP-PRRSV、猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清进行检测。结果，欧洲型PRRSV、美洲型PRRSV、HP-PRRSV阳性血清OD450nm值分别为0.269、1.195和1.274；猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清均小于0.3659（结果见表1-3）。表明本研究建立的方法特异性强。
表1-3 特异性实验
5．1.4 重复性实验
利用优化后的条件建立的ELISA条件进行检测，获得组间变异系数在1.32%～4.97%之间，表明建立的ELISA具有良好的重复性。
5．1.5 临床样品检测
利用建立的ELISA方法对本收集的35份猪血清进行检测，其中美洲型PRRSV阳性29份，阳性率为82.86%（30/35）；且这29份阳性血清在建立的多肽1为抗原包被的间接ELISA中检测也为阳性，阳性符合率为96.67%（29/30）。
6．1 区分经典型PRRSV和HP-PRRSV多肽4建立的间接ELISA：
通过对经典型PRRSV和HP-PRRSV的差异区的非结构蛋白Nsp2（Nsp2）的进行氨基酸序列比对发现，经典型PRRSV和HP-PRRSV存在非结构蛋白Nsp2 的差异“RPMTPLSEPIFLSAPRHKFQQVEEANPAT”。
6.1.1 多肽4的合成
对该表位多肽4经生工生物工程(上海)股份有限公司合成后，合成多肽的纯度＞95%。对合成的多肽溶解成母液（1mg）备用。
6.1.2 ELISA条件的确定
将抗原母液按照终浓度（1000ng、500ng、250ng、125 ng、62.5 ng和31.25 ng），将一抗（猪血清）按照（1:10、1:20、1:40、1:80、1:160和1:320）进行稀释，HRP标记的山羊抗猪酶标二抗按照说明书推荐的1:5000稀释。优化后的ELISA条件为，包被多肽4 250ng、血清稀释度为1：80、酶标二抗浓度1：5000即可获得最大化P/N值。对30份进行检测后计算得到OD450nm值的平均数（X）=0.2297、标准方差（SD）=0.0294，得到本研究的临界值（C）=0.3179。即本实验建立的ELISA对临床样品进行检测OD450nm值≧0.3179判为阳性，其他均判为阴性。
需要指出的是，多肽4建立的间接ELISA检测经典型PRRSV为阳性，检测HP-PRRSV为阴性。
6．1.3 特异性实验
利用优化后的条件，对欧洲型PRRSV、美洲型PRRSV、HP-PRRSV、猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清进行检测。结果，欧洲型PRRSV、美洲型PRRSV、HP-PRRSV阳性血清OD450nm值分别为0.247、0.983和0.252；猪伪狂犬病毒（PRV）、猪圆环病毒（PCV2）、猪瘟病毒（CSFV）、猪流行性乙型脑炎病毒（JEV）高免血清和猪阴性血清均小于0.3179（结果见表1-4）。表明本研究建立的方法特异性强。
表1-4 特异性实验
6．1.4 重复性实验
利用优化后的条件建立的ELISA条件进行检测，获得组间变异系数在1.12%～4.87%之间，表明建立的ELISA具有良好的重复性。
6．1.5 临床样品检测
利用建立的ELISA方法对本收集的35份猪血清进行检测，其中阳性样品4份，阳性率为11.43%（4/35）；且这4份样品在多肽1建立的间接ELISA检测均为阳性，阳性符合率为100%；且这4份样品在多肽3建立的间接ELISA检测均为阳性，阳性符合率为100%。
7 临床检测样品PRRSV感染类型的确定
利用建立的多肽1-ELISA方法对本收集的35份猪血清进行检测，其中阳性32份，阳性-率为91.42%（32/35），表明35份样品中PRRSV阳性32份（指的是美洲型PRRSV、欧洲型PRR=SV和HP-PRRSV）。利用建立的多肽2--ELISA方法对本收集的35份猪血清进行检测，其中欧洲型PRRSV阳性2份，阳性率为5.71%（2/35）；且这2份阳性血清在建立的多肽1为抗原包被的间接ELISA中检测也为阳性，阳性符合率为100%；即35份中欧洲型PRRSV阳性2份；剩下的30份均为美洲型PRRSV（包含有HP-PRRSV）。利用建立的多肽3--ELISA方法对本收集的35份猪血清进行检测，其中美洲型PRRSV阳性29份，阳性率为82.86%（30/35）；且这29份阳性血清在建立的多肽1为抗原包被的间接ELISA中检测也为阳性，阳性符合率为96.67%（29/30）；仅1份样品可疑，阳性符合率符合鉴别试剂盒的要求（95%以上）。利用建立的多肽4--ELISA方法（多肽4建立的间接ELISA检测经典型PRRSV为阳性，检测HP-PRRSV为阴性。）对本收集的35份猪血清进行检测，其中阳性样品4份，阳性率为11.43%（4/35）；且这4份样品在多肽1建立的间接ELISA检测均为阳性，阳性符合率为100%；且这4份样品在多肽3建立的间接ELISA检测均为阳性，阳性符合率为100%；即35份样品中经典美洲型PRRSV阳性4份，HP-PRRSV阳性25份（29-4=25）。根据建立的多肽1--ELISA方法、多肽2--ELISA方法、多肽3--ELISA方法和多肽4--ELISA方法检测35份血清结果为，欧洲型PRRSV阳性2份；经典美洲型PRRSV4份；HP-PRRSV阳性25份。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建省农业科学院畜牧兽医研究所
<120> 猪繁殖与呼吸综合征血清学鉴别诊断试剂盒
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<170> PatentIn version 3.3
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Claims (1)
1.猪繁殖与呼吸综合征血清学鉴别诊断试剂盒，其特征在于：所述试剂盒中含有4条抗原多肽，其序列为：多肽1：PEKPHFPLATEDDVRHH；多肽2 ：LRPHGVSAAQEEIPFGLSSQ；多肽3：EGHLIDLKRV；多肽4：RPMTPLSEPIFLSAPRHKFQQVEEANPAT。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259141A (zh) * | 1997-05-06 | 2000-07-05 | 农业技术研究基金会 | 用于疫苗或诊断测试中的鉴定prrs病毒的肽序列的prrsv抗原位点 |
| CN1458280A (zh) * | 2003-06-02 | 2003-11-26 | 中国农业科学院哈尔滨兽医研究所 | 两个人工合成prrs病毒多表位串联核苷酸序列及应用 |
| CN102175861A (zh) * | 2010-12-23 | 2011-09-07 | 中国动物疫病预防控制中心 | 欧洲型、美洲型猪繁殖与呼吸综合征病毒抗体的间接elisa检测方法 |
| CN102297969A (zh) * | 2011-05-25 | 2011-12-28 | 福建省农业科学院畜牧兽医研究所 | 一种鉴别诊断传统美洲型和变异型猪繁殖与呼吸综合征的免疫荧光试剂的制备方法 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259141A (zh) * | 1997-05-06 | 2000-07-05 | 农业技术研究基金会 | 用于疫苗或诊断测试中的鉴定prrs病毒的肽序列的prrsv抗原位点 |
| CN1458280A (zh) * | 2003-06-02 | 2003-11-26 | 中国农业科学院哈尔滨兽医研究所 | 两个人工合成prrs病毒多表位串联核苷酸序列及应用 |
| CN102175861A (zh) * | 2010-12-23 | 2011-09-07 | 中国动物疫病预防控制中心 | 欧洲型、美洲型猪繁殖与呼吸综合征病毒抗体的间接elisa检测方法 |
| CN102297969A (zh) * | 2011-05-25 | 2011-12-28 | 福建省农业科学院畜牧兽医研究所 | 一种鉴别诊断传统美洲型和变异型猪繁殖与呼吸综合征的免疫荧光试剂的制备方法 |
Non-Patent Citations (3)
| Title |
|---|
| Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey;Carl A. Gagnon,et al;《J Vet Diagn Invest》;20081231;第20卷;545–558 * |
| 多重实时荧光定量PCR鉴别欧洲型、美洲型和高致病性PRRSV检测方法的建立;温青娜 等;《动物医学进展》;20151231;第36卷(第6期);1-8 * |
| 高致病性与传统型PRRSV鉴别诊断间接ELISA方法的建立及初步应用;肖一红 等;《中国畜牧兽医学会畜牧兽医生物技术学分值暨中国免疫学会兽医免疫分会第八次学术研讨会论文集》;20100801;157-161 * |
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