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根据已发表的口蹄疫病毒(FMDV)基因序列设计了一对引物,扩增FMDV 3D后1/3区段,应用反转录聚合酶链式反应(RT-PCR)技术,从组织培养液中扩增出大小为489 bp的基因片段,建立了FMDV RT-PCR检测方法.该方法灵敏度高,可检测出0.01个TCID50的病毒含量;特异性好,不与其他病毒如PRV、PRRSV、JEV等发生交叉反应.阳性样品的检测结果与反向间接血凝(IHA)检测结果的符合率为100%.应用所建立的RT-PCR方法,对采自3个奶牛场的55份鲜奶样品及40份牛鼻拭子样品进行检测.结果表明,所建立的检测方法可用于奶牛隐性感染FMDV的检测. 相似文献
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利用单克隆抗体技术制备抗口蹄疫病毒的单克隆抗体,特异性试验表明其只与O、A、Asia 1型3种血清型FMDV抗原结合。进而采用胶体金标记技术,以胶体金标记的抗口蹄疫病毒单克隆抗体、多克隆血清抗体和葡萄球菌A蛋白为主要材料,研制口蹄疫快速检测试纸条。该试纸条检测O、A、Asia 1型3种血清型灭活口蹄疫病毒均为阳性,检测水疱性口炎病毒、猪水疱病病毒、蓝舌病病毒、猪蓝耳病病毒4种灭活抗原及小反刍兽疫病毒疫苗株均为阴性,试验结果与口蹄疫实时荧光定量RT-PCR方法的完全一致,表明其具有良好的特异性。敏感性试验结果是,试纸条的检测极限为1∶160稀释的样品,其敏感性相当于实时荧光定量RT-PCR方法的1/64。由于试纸条具有操作方便、检测快速等优点,因此该试纸条可以用于大量临床样品的快速检测和现场检测。 相似文献
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光生物素标记的口蹄疫病毒cDNA探针的制备及其对口蹄疫病毒RNA检测的试验研究 总被引：1，自引：0，他引：1
本文首次报道了利用pF1034质粒制备FMDV O型特异性探针,并通过PCR反应扩增FMDO_1K株病毒基因组的第2962位与3071位之间共110bp序列,制备了能检测O型、A型和亚洲I型FMDV RNA的群(组)特异性探针。用硝酸纤维素膜斑点杂交试验表明,二者均能检测出10pg水平的O_9K毒株的纯RNA;但前者只与O型FMDV RNA杂交,与A型及亚洲I型FMDV RNA无交叉杂交现象;而后者则能与O型、A型和亚洲I型的FMDV RNA发生杂交反应。对照试验显示:此两种探针与SVDV ssRNA、BTV dsRNA、EHDV dsRNA、DHV ssRNA、PRV DNA、乳鼠组织细胞RNA、BHK_(21)克隆13细胞RNA及DNA等均不出现交叉杂交现象,但与IBR DNA有假阳性杂交反应。 相似文献
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用荧光定量RT-PCR方法检测猪瘟病毒 总被引：4，自引：1，他引：4
为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10~(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。 相似文献
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R F Meyer C C Brown T W Molitor V N Vakharia 《Journal of veterinary diagnostic investigation》1989,1(4):329-332
Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation. 相似文献
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HeLa细胞Ⅰ型干扰素效应因子实时荧光定量RT-PCR检测方法的建立及初步应用 总被引：1，自引：1，他引：0
本试验旨在建立一种用SYBR GreenⅠ荧光染料检测HeLa细胞Ⅰ型干扰素效应因子ISG15、ISG56、Mx1、OAS和PKR mRNA表达水平的实时荧光定量RT-PCR检测方法,并在口蹄疫病毒(FMDV)L蛋白抑制Ⅰ型IFN发挥效应的信号通路中进行初步应用。利用TRIzol法提取总RNA,经Oligo d(T)15进行反转录,利用PCR扩增各段目的基因,并克隆至pMD18-T载体,转化大肠杆菌DH5α,经鉴定为阳性的重组质粒作为标准品模板建立SYBR GreenⅠ荧光定量RT-PCR标准曲线和熔解曲线,并进行灵敏性、特异性和重复性试验。根据建立的实时荧光定量RT-PCR方法,检测FMDV L蛋白对Ⅰ型IFN效应因子的抑制效果。HeLa细胞在转染FMDV L蛋白真核表达质粒,并受到Ⅰ型IFN刺激后ISG15、ISG56、Mx1、OAS和PKR的相对表达量较转染空载体或表达GST的真核表达质粒明显降低。本试验建立了HeLa细胞Ⅰ型IFN效应因子的实时荧光定量RT-PCR检测方法,为在mRNA水平上对HeLa细胞Ⅰ型IFN效应因子的定量分析奠定了基础,并成功地初步应用于FMDV L蛋白抑制Ⅰ型IFN发挥效应的信号通路的研究中。 相似文献
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A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV. 相似文献
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Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus 总被引：1，自引：0，他引：1
Nordengrahn A Gustafsdottir SM Ebert K Reid SM King DP Ferris NP Brocchi E Grazioli S Landegren U Merza M 《Veterinary microbiology》2008,127(3-4):227-236
A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis. 相似文献
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应用实时荧光定量TaqMan RT-PCR检测口蹄疫病毒 总被引：11，自引：0，他引：11
按照口蹄疫病毒(FMDV)聚合酶3D基因序列,设计合成了引物和探针,经各反应务件的优化,建立了实时荧光定量RT-PcR技术,对细胞培养物、水泡液、水泡皮及分泌物、血液中的FMDV进行了特异性检测和敏感性试验。结果,用300nmol／L的引物浓度和200nmol／L探针浓度,获得的CT值较小,而△Rn最大;可检测到相当于9．1TCID50的病毒RNA;与VSV和其他水泡性病毒不发生交叉反应;制作的标准曲线中各浓度范围内有极好的线性关系,且线性范围宽,相关系数为0．984;组内和组间试验重复性的变异系数(CV)分别为5．4％和6．7％;与常规RT-PCR相比较,该方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点。 相似文献
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OBJECTIVE: To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia. 相似文献
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Dalla Valle L Toffolo V Lamprecht M Maltese C Bovo G Belvedere P Colombo L 《Veterinary microbiology》2005,110(3-4):167-179
The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 101 to 108 copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2 > 0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus. 相似文献
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To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population. 相似文献
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参考GenBank中各个血清型口蹄疫病毒3D、vp1、2A基因的标准序列，设计引物P1／P2和S1／S2。建立用于检测口蹄疫病毒及其利用引物S1／S2克隆片段同源性比较而确定血清型的RT-PCR方法。通过敏感性试验检测，2对引物均可以检测到10TCID50的病毒量；特异性试验的检测，2对引物对正常细胞、牛黏膜病病毒、猪瘟病毒、水疱性口炎病毒、牛传染性鼻气管炎病毒的检测结果均为阴性。利用该方法对病牛的流涎液体、水疱液体、舌皮组织、感染犊牛心脏等组织进行检测初步结果显示：该方法可以对O型和AsiaⅠ型口蹄疫病毒进行特异性检测，能够用于口蹄疫急性及亚临床感染的诊断及流行病学调查。 相似文献