Establishment and Preliminary Application of Nested PCR for the Detection of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) caused significant economic losses of swine industry,it's very important to establish a economy and rapid detection method.To strengthen the prevention and control of porcine circovirus disease,rapid and high sesitive detection method of PCV2 was established using nested PCR.According to PCV2 ORF2 gene conservative region,we designed two pairs of nested PCR primers,constructed ORF2 gene recombinant plasmid for standard template and optimizated the PCR reaction conditions to establish nested PCR detection method of PCV2 and detect the clinical samples of pig farms.Our results showed that the sensitivity and repeatability of this method were very high,the minimum detection level was 10 copies/μL and detection rate of clinical samples was 97.3%.In brief,the established nested PCR detection method of PCV2 in this study provided powerful means for quick and efficient detection of PCV2,and prevention and control of porcine circovirus disease in the swine industry.     

日本脑炎病毒套式RT-PCR方法的建立与初步应用

根据GenBank上登录的日本脑炎病毒(JEV)E基因序列,设计内外2对引物.以JEV疫苗株为模板,建立了检测猪JEV的套式RT-PCR方法.应用该方法对JEV疫苗株RNA进行扩增,获得与预期大小相符,长度为228 bp的目的片段;检出JEV-RNA的灵敏度约为0.3 pg.表明所建立的套式RT-PCR方法对JEV的检测敏感性高、特异性强.     

单管套式PCR检测外源性禽白血病病毒

从贵州省4个养鸡场采集出现肿瘤病变或疑似肿瘤病料样本17份,以单管套式PCR方法检测禽白血病病毒长末端重复序列(LTR),15份病料均扩增出一条213 bp的DNA带,与预期扩增长度相符合,外源性禽白血病病毒的检出率达到88.2%.同时针对禽白血病病毒的囊膜糖蛋白基因(gp85)进行PCR扩增,在被检的17份病料中,7份检出J亚群特异性的DNA带(545 bp),占41.1%;9份检出A亚群特异性的DNA带(691 bp),占52.9%;其中2份病料同时检出J亚群与A亚群特异性的病原核酸.A、J亚群的PCR检出率低于LTR的套式PCR扩增.     

鸡大肠杆菌O2/O74混合血清型菌株iss基因的克隆与序列测定

采用PCR方法对1株O2/O74混合血清型鸡大肠杆菌检测其iss基因的存在,并与1株O2血清型鸡大肠杆菌相比较.结果表明:鸡E.coli O2/O74混合血清型菌株携带iss基因,但采用目前通用的PCR方法并不能检测出iss基因,必须经套式PCR扩增才能检测出iss基因.O2/O74混合血清型菌株iss基因序列与已知禽大肠杆菌iss基因序列同源性为99.7%;与人源大肠杆菌iss基因进行比对,同源性为90.6%;与λ噬菌体bor 基因序列进行比对,同源性为88%,显示了此基因的保守性.     

猪圆环病毒2型巢式PCR检测方法的建立与初步应用

猪圆环病毒2型(porcine circovirus type 2,PCV2)给养猪业造成了重大的经济损失,建立经济、快速的检测方法尤为重要。本研究旨在利用巢式PCR技术,建立高灵敏度PCV2检测平台,以加强对猪圆环病毒病的防控。根据PCV2 ORF2基因保守区,设计内、外2对巢式PCR引物,通过优化PCR反应条件,建立巢式PCR检测方法,并对猪场临床样本进行检测。结果显示,本研究建立的巢式PCR检测方法灵敏度高、重复性强,最低检出量为10拷贝/μL,检出率达97.3%。本研究建立的巢式PCR检测方法为快速、高效地检测PCV2以及为猪圆环病毒病的防控提供有力手段。     

菠菜中3种新烟碱类杀虫剂多残留分析方法研究

寄生隐丛赤壳菌是引起板栗疫病的致病菌。为建立该菌的分子检测技术,本研究首先采用通用引物 ITS1／ITS4对分离自四川雅安、泸州及重庆的寄生隐丛赤壳菌及其他参试菌株的 ITS 区进行 PCR 扩增和测序比对。根据该片段与 GenBank 中隐丛赤壳属其他种的 ITS 序列差异,设计了寄生隐丛赤壳菌的特异性引物 ITSP1／ITSP2,片段扩增大小为462 bp。利用该引物对菌株基因组 DNA 进行扩增,可以将寄生隐丛赤壳菌与其他参试菌区分开,检测灵敏度达30 pg。而以引物 ITS1／ITS4和 ITSP1／ITSP2进行的巢氏 PCR,可检测到30 fg 基因组 DNA,其灵敏度较常规 PCR 提高了1000倍。利用巢氏 PCR 检测体系对发病程度不同的组织和携菌组织进行检测,均能快速稳定地检测出寄生隐丛赤壳菌。     

H3亚型禽流感病毒巢式PCR检测方法的建立

为建立一种H3亚型禽流感病毒(AIV)的检测方法,本研究针对H3亚型AIV HA基因保守序列,设计并筛选出2对特异性引物,通过优化反应条件,建立了H3亚型AIV巢式PCR检测方法。对该法进行特异性和敏感性检验,并用该法对96份临床样品进行检测。特异性试验结果表明该法只能检测到H3亚型AIV,对其他常见禽病病原体不扩增;敏感性试验结果表明该巢式PCR对H3亚型AIV检测下限为1×10³拷贝/μL,灵敏度比常规PCR高100倍;96份临床样品检测结果与病毒分离结果一致。本研究所建立的巢式PCR为H3亚型AIV的诊断提供了一种准确、有效的检测方法。     

Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout,Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested‐PCR study

This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E‐stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin‐fixed, paraffin‐embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.     

甘蔗白叶病植原体巢式PCR检测方法的建立及初步应用

甘蔗白叶病(sugarcane white leaf,SCWL)是由植原体引起的重要甘蔗病害[1],广泛分布在印度、泰国等许多国家[1,2].我国甘蔗产区的栽培品种也有SCWL的发生[3].甘蔗是无性繁殖作物,植原体可通过繁殖种苗进行传播,台湾斑纹叶蝉(Matsumuratetlix hiroglyhious)通过咬食感染甘蔗植株的韧皮部可引起该病害[4].