口蹄疫病毒RT-PCR检测与血清型鉴别

针对编码非结构蛋白的3D基因合成一对引物进行口蹄疫病毒RT-PCR扩增,不同血清型病毒的RNA样本均显现一条457bp的目的带,与预期设计的长度相符合。在敏感性试验中,O型、A型和AsiaⅠ型病毒的最小RNA检出量分别为0.8ng、8ng和8ng。根据GenBank发表的口蹄疫病毒VP1和2A基因序列,采用多重RT-PCR鉴别口蹄疫病毒血清型,O型、A型和AsiaⅠ型病毒的特异性扩增片段分别为200bp、340bp和500bp。对9份乳鼠感染病料进行检测,确诊为O血清型口蹄疫病毒感染。     

O型和Asia1型口蹄疫病毒感染RT-PCR鉴别诊断方法的建立

根据GenBank中O型和Asia1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)的vp3、vp1和2A基因序列,并与其它血清型FMDV的对应基因序列进行比较,设计用于扩增O型和Asia1型FMDV vp1基因的特异性引物,建立O型和Asia1型FMDV RT-PCR鉴别诊断方法。本方法首先用通用型引物进行RT-PCR,确定是否为FMDV感染,然后用特异性引物鉴别O型或Asia1型FMDV的感染。用vp1基因序列分析进行符合性试验,验证了该方法所具有的特异性和敏感性。本方法可用于O型和Asia1型FMD的快速诊断及流行病学调查。     

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus

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.     

牛口蹄疫病毒VP2结构蛋白抗体间接ELISA方法的建立

为建立牛口蹄疫(FMD)抗体的检测方法,本研究将口蹄疫病毒(FMDV)的VP2基因,通过pPROEXTM HTb表达载体在大肠杆菌DH5α中表达,获得大小为35ku的重组VP2蛋白(rVP2),western blot证实rVP2可与FMDV5种血清型的牛阳性血清发生特异性反应。以纯化复性的rVP2为抗原建立了FMDVrVP2间接ELISA方法。重复性试验证实批内、批间变异系数均小于10%。特异性交叉试验表明,该抗原不与常见的其他7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫国家牛阴性血清的特异性为100%;检测感染血清敏感性为97.3%;检测O-AsiaⅠ的二价苗免疫牛血清,与4种商品化试剂盒比较,其符合率分别为69.0%、95.0%、90.4%和86.8%。实验结果表明建立的ELISA方法可以用于口蹄疫感染和免疫抗体检测。     

O型口蹄疫病毒VP1基因的克隆及原核表达载体构建

用Trizol提取O型口蹄疫病毒RNA,根据已经公布的O型口蹄疫病毒核苷酸序列,设计合成1对VP1基因的引物,通过RT-PCR扩增出VP1基因,将其克隆至表达载体pET-32a中。经测序表明,目的基因VP1已正确地整合至表达质粒中。     

南非Ⅱ型口蹄疫病毒抗原表位的筛选及抗原性分析

目前,我国南非Ⅱ型(SATⅡ)口蹄疫病毒(FMDV)的防控形势十分严峻,为了防止SATⅡ型FMD的跨境传入,迫切需要建立其特异的检测方法。本研究以SATⅡ型FMDV结构蛋白氨基酸序列为依据,利用分子生物学软件分析了FMDV结构蛋白VP1~VP3上可能的抗原表位,并人工合成了8条表位多肽。通过采用SATⅡ型FM-DV阳性血清进行ELISA反应,检测其反应原性;通过采用与载体蛋白偶联的合成肽免疫小鼠,测定小鼠血清中抗体效价,检测合成肽的免疫原性。结果表明,合成的8条多肽均能与SATⅡ型FMDV阳性血清结合,其中的6条多肽免疫小鼠后能产生针对多肽的抗体。本研究为利用串联表位为抗原检测SATⅡ型FMDV抗体方法的建立奠定了基础。     

口蹄疫病毒结构蛋白基因vp1的表达与应用研究

体外克隆口蹄疫病毒vp1基因，构建重组表达载体pET28a-vp1。将此重组质粒转化到受体菌BL21（DE3）中，进行诱导表达，SDS-PAGE和蛋白质印迹分析表明，诱导5h后表达量达到最高，表达产物大小约为33Ku-40Ku，表达蛋白能与口蹄疫病毒阳性血清产生特异性免疫反应。经HPLC纯化后，以重组蛋白为抗原，建立检测VP1蛋白抗体的ELISA方法，检测猪牛血清样品，免疫抗体检测结果与口蹄疫液相阻断ELISA检测结果呈正相关，能反映出免疫抗体动态变化，对临床样品口蹄疫病毒血清抗体检测，两种方法有一定相关性，但不显著。所以以重组VP1蛋白为检测抗原的ELISA方法有望用于口蹄疫免疫抗体监测。     

口蹄疫病毒RT-PCR检测和分型方法的建立及初步应用

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。     

应用荧光RT-PCR检测亚欧型口蹄疫病毒

根据口蹄疫病毒基因组的5’非翻译区序列比较保守的特点,采用软件设计亚欧型(A、O、C及Asia-1型)FMDV通用的引物和探针,经对反应条件和反应体系进行优化,建立了亚欧型FMDV的实时荧光RT-PCR检测方法。研究表明,该方法检测阳性质粒模板的线性范围为1.9×107-1.9×10拷贝,最低可检测19个拷贝。通过对FMDV各血清型(A、O、C、Asia-1及SAT-1,2,3型)的检测,证实该方法对亚欧型FMDV具有良好的特异性,能有效区分亚欧型和南非型FMDV感染。本研究为亚欧型口蹄疫的快速诊断和流行病学调查提供了新的方法。     

一种用于口蹄疫病毒实时荧光RT-PCR检测的引物和方法

本文建立了一种用于口蹄疫病毒实时荧光RT-PCR检测的引物和方法。分析所有已报道的口蹄疫病毒通用型基因组序列,分别设计引物和荧光探针,通过实时荧光RT-PCR检测,得到口蹄疫通用型的特异性荧光曲线,可以确定血清型,检测结果准确、可靠。     

O型、A型及Asia-1型口蹄疫病毒多重RT-PCR检测方法的建立

根据口蹄疫病毒VP1基因序列,利用Primer Premier 5.0软件设计4条特异性引物,通过对退火温度等反应条件进行优化,建立能够同时扩增出O型、A型、Asia-1型口蹄疫病毒的多重RT-PCR检测方法。结果表明,建立的方法最低可以检测出约含1 pg/μL的病毒样品;该方法对猪瘟病毒、猪细小病毒、猪伪狂犬病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒等其他相关病毒的检测结果均为阴性,特异性良好。建立的方法能够对口蹄疫O型、A型、Asia-1型病毒准确定型,可广泛用于口蹄疫病毒3个血清型的快速检测和分子流行病学调查。     

Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virus

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.     

应用RT-PCR方法快速检测水泡性口炎病毒

针对水泡性口炎病毒(VSV)的两种血清型设计了2对引物，建立RT-PCR方法，用于检测VSV。VSV接种细胞出现明显的细胞病变，经RT-PCR检测为阳性，而检测口蹄疫、猪水泡病均为阴性，说明引物具有较好的特异性。     

Development and Application of Two-temperature RT-PCR for Detectionof Type O Foot and Mouth Disease Virus

According to the gene sequences analysis of foot and mouth disease virus (FMDV) in GenBank,a pair of specific primers was designed in the conserved sequence of type O FMDV P1 gene. The reaction parameters were optimized using the uniform design method to develop a two-temperature RT-PCR method for detection of type O FMDV.The results of sensitivity and specificity showed that the two-temperature RT-PCR method was only specific for type O FMDV without amplification of the other viruses. The amplified fragment was same with the expected length.The cloning and sequencing results revealed that the sequence of amplified fragment had 100% simililarity to the target sequence,and the minimum detection quantity was 1.665 pg/μL,the effective detection rate was consistent with the three step RT-PCR sensitivity test results. 54 taper toxicity test pigs were detected,and positive identification results and three-step PCR results was consistent.Compared with the three-step PCR,it could save 20 min.These results indicated that the developed two-temperature RT-PCR for detection of type O FMDV was a kind of accurate,rapid,specific and sensitive detection method.     

口蹄疫O型病毒二温式RT-PCR检测方法的建立及初步应用

试验通过对口蹄疫病毒核苷酸序列的比对分析,在O型口蹄疫病毒的P1基因保守区,设计1对特异性引物,应用均匀设计法优化反应参数,建立口蹄疫O型病毒二温式RT-PCR检测方法。对该法进行特异性试验、敏感性试验检测。结果表明,该二温式RT-PCR方法只对口蹄疫O型病毒敏感,对其他血清型的口蹄疫病毒及常见的猪病病毒均不敏感;扩增条带与预期目的片段大小相符,扩增片段经克隆、测序发现与引物所在基因序列的同源性为100%;检测病毒RNA的敏感性为1.665 pg/μL,其敏感性与三步法PCR敏感性检测结果没有差异。运用该法对54头攻毒试验的动物进行检测,阳性鉴定结果与三步法PCR鉴定结果一致,与三步法PCR相比该法节省了20 min,表明所建立的口蹄疫O型病毒二温式RT-PCR方法是一种准确、快速、特异、敏感的检测方法。     

Microarray-based detection and typing of foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is the most economically important veterinary pathogen because of its highly infectious nature and the devastating effects the virus has on the livestock industry. Rapid diagnostic methods are needed for detection and typing of FMDV serotypes and differentiation from other viruses causing vesicular diseases. We developed a microarray-based test that uses a FMD DNA chip containing 155 oligonucleotide probes, 35-45 base pair (bp) long, virus-common and serotype-specific, designed from the VP3-VP1-2A region of the genome. A set of two forward primers and one reverse primer were also designed to allow amplification of approximately 1100 bp of target sequences from this region. The amplified target was labelled with Alexa-Fluor 546 dye and applied to the FMD DNA chip. A total of 23 different FMDV strains representing all seven serotypes were detected and typed by the FMD DNA chip. Microarray technology offers a unique capability to identify multiple pathogens in a single chip.     

Development and Primary Application of Indirect ELISA Method for Detecting the IgA Antibody against Foot-and-mouth Disease Virus Type A in Porcine

An indirect ELISA for detecting the IgA antibody against porcine foot-and-mouth disease virus (FMDV) type A was developed by using purified FMDV structural protein VP1 as coating antigen, mouse anti-pig IgA monoclonal antibody as second antibody and HRP-conjugated goat anti-mouse IgG as third antibody.The concentration of coating antigen was optimized as 3.50 μg/mL,the dilution and reaction time of second antibody and third antibody were optimized as 1:10 000 and 30 min, respectively.There was no cross-reactivity with anti-CSFV, PRRSV and other pathogen specific IgA antibodies.The positive detection rate of FMDV type A infectedsamples was above 90%.The coefficient variation of intra-and inter-assay was ranged from 3.16% to 9.76%.The ELISA method described in this study was proved to be specific and rapid for the detection of FMDV specific IgA antibody.It was potential to be applied for detection the level of FMDV specific IgA and evaluate the effect of mucosal immunity.Besides,it provided a new method for clinical diagnosis of foot-and-mouth disease.     

猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

为建立一种检测口蹄疫病毒（foot-and-mouth disease virus,FMDV）特异性IgA抗体的检测方法,本研究以原核表达系统表达纯化的FMDV结构蛋白VP1作为包被抗原,以鼠抗猪IgA单克隆抗体为二抗,辣根过氧化酶标记的羊抗鼠IgG抗体为三抗,建立猪A型FMDV特异性IgA抗体间接ELISA检测方法。确定抗原包被浓度为3.50 μg/mL,二抗与三抗的最佳稀释度为1∶10 000,二抗和三抗作用时间均为30 min。所建立的方法与抗猪瘟病毒、猪繁殖与呼吸道综合征病毒等病原的特异性IgA抗体间无交叉反应,A型口蹄疫感染样品的阳性检出率在90%以上,批内和批间重复性试验的变异系数介于3.16%~9.76%。该方法为监测FMDV特异性IgA抗体水平变化规律及猪的黏膜免疫效果评价及口蹄疫的早期诊断提供了一种新方法。