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1.
WSSV和IHHNV二重实时荧光PCR检测方法的建立   总被引:6,自引:2,他引:4  
根据基因库中对虾白斑综合征病毒WSSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了WSSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对WSSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对WSSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为WSSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为WSSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于WSSV和IHHNV的检测具有特异、敏感、快速、定量等优点。  相似文献   

2.
常规PCR及RT-PCR已用于对虾DNA及RNA病毒检测,但存在费时、灵敏度较低、不能定量等问题。建立了TaqMan实时荧光定量PCR及RT-PCR方法,分别用于检测白斑综合症病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)及桃拉综合征病毒(TSV)、黄头病毒(YHV)4种对虾病毒。与常规PCR及RT-PCR比较,所建立的TaqMan实时荧光定量PCR及RT-PCR检测上述4种对虾病毒不仅有很高的特异性,检测灵敏度也提高了10~100倍,同时还具有快速、简便、不污染环境、重复性好、实时定量等优点,可明显提高对虾病毒检验检疫工作质量及效率。  相似文献   

3.
对虾白斑综合征(WSS)和桃拉病(TS)是两种严重危害当 前对虾养殖业的对虾病毒性疾病。目前对虾病毒病的诊断 手段主要包括病理学和生物学方法、免疫学方法、分子杂交 方法及PCR方法等。其中PCR方法是这些方法中特异 性最强、敏感最高的病原检测手段,在国外已被广泛应用于 对虾病毒病的检测和诊断。多重PCR是一种特殊PCR形 式,其最突出特点,即一次PCR反应,就可同时检测、鉴别出 多种病原体,在临床混合感染的鉴别诊断上具有其独特优势 和很高的实用价值。 本试验建立了二温式多重PCR同时检测鉴别WSSV和 TSV的方法,并用该多重PCR方法对广西沿海地区对虾养殖 业WSSV和TSV感染状况进行了初步调查。现将结果报告 如下。  相似文献   

4.
比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株:即唐海分离株(渤海湾)、宁波分离株(东海),深圳分离株(南海)的同源性。三个WSSV分离株基因组的限制笥内切酶(Sac Ⅰ,HindⅢ,PstⅠ)酶切多态(RFLP)以及病毒结构蛋白图谱完全一致,证实造成我国从南对北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒(RV-PJ-PRDV),斑节对虾白斑综合征杆状病毒(WSBV-PmNOBⅢ)基因组核酸片段特异性引物进行PCR扩增,结果均能从中国一杆状病毒(WSSV)基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒(WSSV)与斑节对虾白斑综合征杆状病毒(WSBV-PmNOBⅢ),日本对虾相状RV-PJ=PRDV)同源率分别为100%与97%,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了依据。  相似文献   

5.
为进一步提高RT-PCR检测西部马脑炎病毒(WEE)病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT-PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。  相似文献   

6.
对虾白斑综合征杆状病毒同源性比较的研究   总被引:4,自引:1,他引:3  
比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株即唐海分离株(渤海湾),宁波分离株(东海),深圳分离株(南海)的同源性。三个WSSV分离株基因组的限制性内切酶(Sac I,Hind III,Pst I)酶切多态(RFLP)以及病毒结构蛋白图谱完全一致,证实造成我国从南至北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒(RV-PJ=PRDV),斑节对虾白斑综合征杆状病毒(WSBV=PmNOBIII)基因组核酸片段特异性引物进行PCR扩增,结果均能从中国对虾白斑杆状病毒(WSSV)基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒(WSSV)与斑节对虾白斑综合征杆状病毒(WSBV=PmNOBIII),日本对虾杆状病毒(RV-PJ=PRDV)同源率分别为100%与97%,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了证据。  相似文献   

7.
建立特异、灵敏的逆转录聚合酶链反应(RT-PCR)技术,结合碱性磷酸酶标记的探针杂交检测鼠肝炎病毒(MHV),采用MHV-3,MHV-A59病毒株感染DBT细胞,37℃培养,待细胞出现病变时收集提取病毒RNA。依据MHV基因序列设计一对高度保守区特异性引物,进行RT-PCR扩增,结果可见147bp的鼠肝炎病毒产物特异扩增带。敏感性实验检测到10pg的鼠肝炎病毒RNA,同时用ELISA方法对照。结果提示应用RT—PCR技术结合探针杂交检测鼠肝炎病毒。具有简便、快速、灵敏等优势。本研究在国内尚未见报道。  相似文献   

8.
目的建立呼肠孤病毒Ⅲ型(Re03)RT—PCR检测方法,应用于实验动物及人用动物源性材料及生物制品外源Re03的检测。方法根据已发表的呼肠孤病毒Ⅲ型(Re03)M1基因序列,设计合成引物。提取Re03细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、重复性试验。结果建立的Re03RT—PCR检测方法特异、敏感、稳定。以Re03RNA逆转录产物为模板,所能检测RNA最小模板浓度为0.42pg/μL,可检测病毒最小滴度为10^-9/mL。结论建立的呼肠孤病毒Ⅲ型(Re03)RT—PCR检测方法可用于实验动物及人用动物源性材料及生物制品外源Re03的检测。  相似文献   

9.
本文建立了一种同时检测猪圆环病毒2型(PCV2)、细小病毒(PPV)、及伪狂犬病毒(PRV)疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp(PCV2)、581bp(PPV)、372bP(PRV gB)及147bp(PRV gE)四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10^-6.2、10^-3.8、10^-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.  相似文献   

10.
RT—PCR法检测我国东南沿海凡纳滨对虾的桃拉综合征病毒   总被引:1,自引:0,他引:1  
桃拉综合征病毒(Taura syndrome virus,TSV)引致养殖及野生对虾急性传染病,14d至40d的幼虾极易感。该病毒属微RNA病毒科(Picornaviridae),近年来研究者认为应归类为类蟋蟀麻痹病毒属(Cricket paralysis—like viruses),因其基因组特征更接近于前所未分类的昆虫单股RNA病毒,如丙型果蝇病毒。  相似文献   

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12.
White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang (93.3%), liuao (66.7%), Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.  相似文献   

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14.
Prevalence of Three Shrimp Viruses in Zhejiang Province in 2008   总被引:2,自引:0,他引:2  
White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province,China,in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations,8 farms were positive for WSSV,8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shr...  相似文献   

15.
The Infectious hypodermal and hematopoietic necrosis virus(IHHNV) and Taura syndrome virus(TSV) are two important shrimp viruses in cultured shrimp in America.These two viruses were transmitted to China at the beginning of the 21st century.In this study,214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection.The results showed that there were a high prevalence of IHHNV(65.42%) and low prevalence of TSV(3.27%) in the tested samples.Several samples were found to be co-infected with these two viruses.A 3 kb fragment of 7 positive IHHNV samples and a structure protein region(ORF2) of three TSV positive samples were amplified and sequenced.The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii.Phylogenetic analysis showed that TSV isolates were clustered into two groups,Asia and America group,which was genetically correlated to geographic distribution.  相似文献   

16.
The Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV) are two important shrimp viruses in cultured shrimp in America. These two viruses were transmitted to China at the beginning of the 21st century. In this study, 214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection. The results showed that there were a high prevalence of IHHNV (65.42%) and low prevalence of TSV (3.27%) in the tested samples. Several samples were found to be co-infected with these two viruses. A 3 kb fragment of 7 positive IHHNV samples and a structure protein region (ORF2) of three TSV positive samples were amplified and sequenced. The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii. Phylogenetic analysis showed that TSV isolates were clustered into two groups, Asia and America group, which was genetically correlated to geographic distribution. Foundation item: This work was supported by 973 project (2006CB101801)  相似文献   

17.
目的:建立一种同时检测鸭圆环病毒(DuCV)和鸭I型肝炎病毒(DHV)病原体的二重PCR技术。方法:根据DuCV和DHV的基因文库,分别设计了2对与DuCV和DHV某段基因序列互补的引物,用这2对引物对同一样品中DuCV和DHV模板进行二重PCR扩增。结果与结论:用建立的方法均同时得到了2条特异性的大小与实验设计相符(DuCV:245bp;DHV:569bp)的二重PCR扩增带,而且对其他禽病病原的PCR扩增结果均为阴性,能同时检出56Pg的DHVRNA模板和6Pg的DuCVDNA模板。  相似文献   

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