猪伪狂犬病病毒PCR诊断方法的建立
摘要
伪狂犬病病毒(Pseudorabies virus,PRV)是引起母猪繁殖障碍的主要病原之一,常常引起母猪流产、死胎;成年猪则系隐性感染,长期带毒排毒,严重影响种猪场生产及优良品种的推广,给养猪业造成极大的损失。疫苗免疫接种和疾病的早期鉴别诊断是有效防制和根除伪狂犬病的根本措施。传统的病毒分离、血清学方法等检测技术在猪伪狂犬病防制中发挥了重要作用,但这些方法存在费时、费力、操作繁琐、敏感性不高或特异性不强、不能检出潜伏感染和对病毒进行定量检测等缺点。因此,探求新的PRV检测技术是伪狂犬病防制研究的主要内容之一。
gH基因是伪狂犬病病毒中较保守和病毒复制所必需的基因;gE基因是伪狂犬病病毒的毒力基因也是多种基因缺失疫苗的缺失基因。本研究设计并合成了PRV的gH和gE基因序列的2对引物,分别建立了gH、gE的单一PCR检测方法,并用琼脂糖电泳鉴定PCR产物,回收PCR产物测序,与预期的结果完全相符,证实了该扩增片段的特异性,并对PPV DNA、PCV DNA和正常PK-15细胞基因组DNA的PCR扩增均为阴性,证明了PCR检测方法的特异性结果证明所扩增的产物均为特异性的核酸片段。对不同核酸提取方法进行了比较,发现异硫氰酸胍一步裂解法费用低、省时、重复性好。同时,在单一PCR方法的基础上,进行引物浓度、退火温度等反应条件的优化后,建立了gH和gE的双重PCR检测方法,可以在一个PCR反应中同时扩增出gH和gE基因两种不同大小的特异性片段。分别提取gH和gE基因的DNA,并测其含量,将病毒核酸连续作10倍的梯度稀释,单一PCR方法最低可检出0.48pggH和0.6pg gE基因的病毒核酸;双重PCR方法最低可同时检出4.8pg gH和6pg gE基因的的病毒核酸。本研究建立的复合PCR方法,可以在24h内对样品进行快速检测并且能够鉴别野毒和疫苗毒感染。
本研究在伪狂犬病病毒gH和gE基因区域内分别设计两对引物和一对探针,建立了定量和鉴别伪狂犬病病毒野毒株与疫苗株的荧光定量PCR检测方法。结果表明Taqman PCR灵敏度达10~1拷贝数量级,线性范围为10~7-10~0copiesl/reaction,达8个数量级,其灵敏度明显高于普通PCR。用此方法对18份疑似组织样品进行检测,结果和血清中和试验检测结果基本一致。此方法具有快速、灵敏、特异、重复性好和能对样品进行批量定量检测等优点,并且以闭管的模式操作,减少了以后步骤污染的可能性,整个PCR检测过程少于2个小时。本研究的建立,为猪伪狂犬病病毒的早期诊断、鉴别诊断和定量分析猪伪狂犬病毒感染程度奠定了基础。
Pseudorabies virus(PRV)is one of the most important pathogens responsible for reproductive failure,including embryonic death,stillbirth and which acutely threaten the breeding.Severity influence swine field production and fineness varietal generalization,pose galactic casualty.The vaccine immunifaction and the disease early diagnosis is the basic measure of preventing and eradicating Pseudorabies.Conventional virus isolation and serological method are time consuming and are comparatively low in sensitivity.These methods detection infection and quantitaly detect virus and so on.Therefor,the new method of prevention and detection for PRV is in urgently needed.
gE gene is the pecant gene of PRV and being the deleted gene of gene deleted vaccines.In this study,we design and conbine two pairs of primers gH and gE gene,and establish the single PCR of gH and gE gene respectively.The results certificate that amplification productions are specific nucleic acid fragments after identifying production of PCR through agarose electrophoresis.we discover guanidinium isothiocyanate schizolysis assay is cheaper,save time and good reproducibility through comparing different nucleic acid extraction methods.Based on the single PCR assay,primer concentration,renaturation temperature were optimized,a duplex PCR system for both of them in one tube was established to distinguish the two genes.we extract DNA of gH and gE gene,measure contents and multiproportion attenuate of 10 times viral nucleic acid,the single PCR was capable of detecting target viruses at minimum 0.48pg(gH)and0.6pg(gE)and this established duplex PCR assay was capable of detecting target viruses at minimum 4.8 pg(gH) and 6pg(gE),the duplex PCR can quickly detect and identify infection which was caused by campus bane or vaccine in 24 h.
The method contains two pairs of primers of gH,gE gene each and two internal dual-labeled fluorogenic probe.We use Flurogenic quantitative PCR established a rapid and reproducible method for assessment of PRV loads in animals and products.The sensitivity of the assay was 10~1 copies per reaction.The assay is linear within 8-log dynamic range.The sensitivity was obviously higher than converntional PCR.The assay was used to detected 18 doubtful tissue samples which had the completely coincidence with Serum neutralization.The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2h or less.The construction of real-time quantitative PCR provides the basis for the early and rapid detection and analysising the infect degree of PRV.
gH基因是伪狂犬病病毒中较保守和病毒复制所必需的基因;gE基因是伪狂犬病病毒的毒力基因也是多种基因缺失疫苗的缺失基因。本研究设计并合成了PRV的gH和gE基因序列的2对引物,分别建立了gH、gE的单一PCR检测方法,并用琼脂糖电泳鉴定PCR产物,回收PCR产物测序,与预期的结果完全相符,证实了该扩增片段的特异性,并对PPV DNA、PCV DNA和正常PK-15细胞基因组DNA的PCR扩增均为阴性,证明了PCR检测方法的特异性结果证明所扩增的产物均为特异性的核酸片段。对不同核酸提取方法进行了比较,发现异硫氰酸胍一步裂解法费用低、省时、重复性好。同时,在单一PCR方法的基础上,进行引物浓度、退火温度等反应条件的优化后,建立了gH和gE的双重PCR检测方法,可以在一个PCR反应中同时扩增出gH和gE基因两种不同大小的特异性片段。分别提取gH和gE基因的DNA,并测其含量,将病毒核酸连续作10倍的梯度稀释,单一PCR方法最低可检出0.48pggH和0.6pg gE基因的病毒核酸;双重PCR方法最低可同时检出4.8pg gH和6pg gE基因的的病毒核酸。本研究建立的复合PCR方法,可以在24h内对样品进行快速检测并且能够鉴别野毒和疫苗毒感染。
本研究在伪狂犬病病毒gH和gE基因区域内分别设计两对引物和一对探针,建立了定量和鉴别伪狂犬病病毒野毒株与疫苗株的荧光定量PCR检测方法。结果表明Taqman PCR灵敏度达10~1拷贝数量级,线性范围为10~7-10~0copiesl/reaction,达8个数量级,其灵敏度明显高于普通PCR。用此方法对18份疑似组织样品进行检测,结果和血清中和试验检测结果基本一致。此方法具有快速、灵敏、特异、重复性好和能对样品进行批量定量检测等优点,并且以闭管的模式操作,减少了以后步骤污染的可能性,整个PCR检测过程少于2个小时。本研究的建立,为猪伪狂犬病病毒的早期诊断、鉴别诊断和定量分析猪伪狂犬病毒感染程度奠定了基础。
Pseudorabies virus(PRV)is one of the most important pathogens responsible for reproductive failure,including embryonic death,stillbirth and which acutely threaten the breeding.Severity influence swine field production and fineness varietal generalization,pose galactic casualty.The vaccine immunifaction and the disease early diagnosis is the basic measure of preventing and eradicating Pseudorabies.Conventional virus isolation and serological method are time consuming and are comparatively low in sensitivity.These methods detection infection and quantitaly detect virus and so on.Therefor,the new method of prevention and detection for PRV is in urgently needed.
gE gene is the pecant gene of PRV and being the deleted gene of gene deleted vaccines.In this study,we design and conbine two pairs of primers gH and gE gene,and establish the single PCR of gH and gE gene respectively.The results certificate that amplification productions are specific nucleic acid fragments after identifying production of PCR through agarose electrophoresis.we discover guanidinium isothiocyanate schizolysis assay is cheaper,save time and good reproducibility through comparing different nucleic acid extraction methods.Based on the single PCR assay,primer concentration,renaturation temperature were optimized,a duplex PCR system for both of them in one tube was established to distinguish the two genes.we extract DNA of gH and gE gene,measure contents and multiproportion attenuate of 10 times viral nucleic acid,the single PCR was capable of detecting target viruses at minimum 0.48pg(gH)and0.6pg(gE)and this established duplex PCR assay was capable of detecting target viruses at minimum 4.8 pg(gH) and 6pg(gE),the duplex PCR can quickly detect and identify infection which was caused by campus bane or vaccine in 24 h.
The method contains two pairs of primers of gH,gE gene each and two internal dual-labeled fluorogenic probe.We use Flurogenic quantitative PCR established a rapid and reproducible method for assessment of PRV loads in animals and products.The sensitivity of the assay was 10~1 copies per reaction.The assay is linear within 8-log dynamic range.The sensitivity was obviously higher than converntional PCR.The assay was used to detected 18 doubtful tissue samples which had the completely coincidence with Serum neutralization.The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2h or less.The construction of real-time quantitative PCR provides the basis for the early and rapid detection and analysising the infect degree of PRV.
引文
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