建立基于TaqMan探针的李坏死环斑病毒实时荧光RT-PCR检测方法

李坏死环斑病毒(Prunus necrotic ringspot virus, PNRSV)是世界部分范围内分布的有害生物, 亦是我国重点关注的检疫对象。根据PNRSV各株系衣壳蛋白基因的保守序列, 设计特异性引物和TaqMan荧光探针, 进行了探针、引物和Mg2+浓度等反应体系和条件的优化实验, 确定最佳的引物浓度为400 nmol/L、探针浓度为333 nmol/L、Mg2+离子浓度为5 mmol/L和dNTPs浓度为0.43 mmol/L时, 其灵敏度达23个拷贝数。利用建立的实时荧光RT-PCR检测方法对PNRSV樱桃分离物进行了成功检测。这个方法具有灵敏、准确、简便、快速的特点, 适合于李坏死环斑病毒的检测和鉴定。     

利用异种动物抗血清双抗夹心法检测小麦黄花叶病毒

小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV),属于马铃薯Y病毒科(Potyviridae),大麦黄花叶病毒属(Bymovirus),传播介体为禾谷多粘菌(Polymyxa graminis),与发生在欧美的小麦梭条花叶病毒(WSSMV)为同一属内的两种病毒。该病毒在我国分布广泛,在长江流域各省份以及济南、陕西等都有分布,对小麦生长.发育构成严重危害。一般可引起小麦减产10％～30％,严重时达70％,甚至绝产。以往对该病害的诊断主要是根据田间的症状表现,有时很难与由其他病原或环境因子引致症状相区分,目前,关于WYMV的问接酶     

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

流式微球一步法快速免疫检测马铃薯A病毒

[目的]建立流式微球一步法快速免疫检测马铃薯A病毒(PVA)的新方法.[方法]以荧光微球为反应载体,通过在微球表面进行双抗夹心免疫反应形成微球-捕获抗体-PVA-标记FITC检测抗体的复合物,利用流式细胞仪荧光检测系统收集荧光信号.[结果]通过实验优化检测条件,最佳捕获抗体工作浓度为4μg/mL、最佳检测抗体工作浓度为1:25倍稀释、最佳反应时间为2h;与马铃薯Y病毒、莴苣花叶病毒、番茄环斑病毒等均未出现交叉反应;阳性样品经64倍稀释后依然可检出,检测灵敏度是传统微孔板ELISA的4倍.[结论]流式微球一步法能灵敏、快速、简便的检测马铃薯A病毒.     

Time-resolved fluoroimmunoassay as a method for detection of Erwinia chrysanthemi in potato peel extracts

J.M. VAN DER WOLF. 1993. Time-resolved fluoroimmunoassay (TR-FIA) was compared with double antibody sandwich (DAS)-ELISA for detection of Erwinia chrysanthemi antigens in potato peel extracts. Pure cultures were used to optimize TR-FIA with respect to the microplate washing procedure and dilution buffer compositions.
The detection threshold level for spiked potato peel extracts with the optimized TR-FIA format was 10⁵ cells ml^-1 as for the detection level of DAS-ELISA. The signal to background ratios at concentrations above 10⁵ cells ml^-1 were higher with TR-FIA than with DAS-ELISA. The dynamic range of TR-FIA was also superior to that of DAS-ELISA.
It can be concluded that TR-FIA is an attractive alternative to DAS-ELISA as a detection method for Erw. chrysanthemi, especially when quantification is required.     

Quantification of intact 146S foot-and-mouth disease antigen for vaccine production by a double antibody sandwich ELISA using monoclonal antibodies

A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146S antigen of foot-and-mouth disease virus (FMDV) strain A10 Holland grown in suspension cultures of surviving bovine tongue epithelium. When virus harvests were incubated with trypsin--which affects VP1, the most immunogenic structural protein of FMDV--the concentration of 146S antigen as determined by ELISA was reduced by greater than 90%. Therefore, the test detected essentially only those virus particles with intact VP1. When the test was compared with the sucrose density gradient method, concentrations of 146S antigen correlated well (r = 0.87). The rate of variation in both tests was the same. In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146S antigen in many samples, and also indicates when VP1 of 146S particles has disintegrated by the action of proteases.     

Detection of sugarcane yellow leaf virus,the causal agent of yellow leaf syndrome in sugarcane by DAS-ELISA

DAS-ELISA studies were conducted on detection of sugarcane yellow leaf virus (SCYLV) causing yellow leaf syndrome (YLS) of sugarcane in leaf and juice antigens. Among the two types of antigen sources used for the virus detection, juice antigen showed high titre for the virus as compared to leaf antigen. Assay with juice samples recorded more number of varieties positive to the virus. Further DAS-ELISA studies revealed that plants raised from disease-infected planting materials recorded high titre for SCYLV as compared to those raised from symptom-free seed canes. Similarly, assaying SCYLV titre in plant and ratoon crop in the field showed that SCYLV infection was partial in plant crop and in the subsequent ratoon crop, all the samples were positive to the virus. ELISA studies also indicated that 33 of 41 cane varieties showing YLS were positive to the virus.     

鼻咽癌患者血清中抗Epstein-Barr病毒早期和晚期膜抗原抗体的检测

对Epstein-Barr(EB)病毒抗原的研究,发现有淋巴细胞确定的膜抗原(Lydma)、早期抗原(EA)、壳抗原(VCA)、核抗原(EBNA)、早期膜抗原(EMA)和晚期膜抗原(LMA)。除了Lydma抗原外,鼻咽癌患者对上述抗原均产生相应的IgG和IgA抗体。因而研究这些抗体,对阐明EB病毒与鼻咽癌的关系及鼻咽癌的早期诊断都十分有价值。     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.     

烟草环斑病毒IC-RT-Realtime PCR检测方法研究

本研究首次应用免疫捕捉反转录实时荧光PCR技术(IC-RT-RealtimePCR)检测烟草环斑病毒,由于综合运用了抗原抗体特异性结合、核酸分子杂交、高灵敏度实时荧光PCR技术的优点,从而使病毒检测在特异性、灵敏度、稳定性等技术指标上比传统的DAS-ELISA方法都有显著提高,解决了烟草环斑病毒检测工作中由于隐症、病毒浓度低、干扰物质存在而影响检测结果的问题。     

Virus-vector relationships of chickpea chlorotic dwarf geminivirus and the leafhopper Orosius orientalis (Hemiptera: Cicadellidae)

Chickpea chlorotic dwarf geminivirus (CCDV) is one of the viruses associated with chickpea stunt disease. It is transmitted by the leafhopper Orosius orientalis. The minimum acquisition access period (AAPmin) and inoculation access period (IAPmin) were found to be less than 2 min, while the minimum latency period (LPmin) was less than 2 h. The median AAP, IAP and LP were 8.0 h, 2.3 h and 27.7 h, respectively. No difference in transmission rates (proportion of leafhoppers able to transmit) was observed between male and female leafhop-pers. In serial transmission experiments, transmission was shown to be persistent, and after a 2-day AAP about 80% of the leafhoppers transmitted the virus for most of their life. The virus could be detected in individual leafhoppers by DAS-ELISA. It did not multiply in the leafhopper, but, instead, decreased in concentration during leafhopper feeding on a non-host of the virus.     

Prunus necrotic ringspot virus isolates in stone fruit germplasm accessions and cultivars in Israel

Prunus necrotic ringspot virus (PNRSV) was detected in almonds, plum and apricot germplasm accessions and local almond cultivars in Israel. PNRSV was widespread both in wild and cultivated almond trees and uncommon in wild apricots and plums. The possible variation among the PNRSV isolates was initially evaluated by restriction analysis of PCR products representing the CP gene with the endonuclease RsaI and followed by nucleotide sequence analysis of selected isolates. It was concluded that all 13 isolates belong to group PV96, the largest cluster of PNRSV isolates, described previously. Two PNRSV isolates, one from a plum accession and one from an almond cultivar, were found to be distinct members of group PV96 with unique nucleotide modifications not found in other documented isolates of this virus. However, no PNRSV isolate typical to a specific host and/or to the Middle East region could be identified. This study expands the body of data on variability of PNRSV isolates and highlights the importance of assessing the virus status of germplasm collections by applying reliable diagnostic and differentiating methods.     

犬瘟热病毒（CDV）ELISA检测方法的建立与应用

目的建立犬CDV抗体ELISA检测方法。方法培养vero细胞,接种CDV病毒,制备vero正常抗原和CDV特异抗原,滴定酶结合物和抗原最佳工作浓度,并进行精密性、敏感性、稳定性、特异性实验。结果正常、特异抗原和酶结合物最佳工作浓度分别为1∶32 000、10μg/mL和1∶8 000;正常、特异抗原批内变异系数分别为9.1%和5.8%,批间平均变异系数分别为8.8%和6.6%;检测灵敏度为1∶2 560;与犬细小病毒（CPV）、犬肝炎病毒（ICHV）均无交叉反应。稳定性试验相对偏差小于25%。结论建立的ELISA方法重复性、稳定性好,特异性、敏感性强。可用于犬CDV抗体的检测。     

Development and Evaluation of a DAS-ELISA for Rapid Detection of Tembusu Virus Using Monoclonal Antibodies against the Envelope Protein

Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.     

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Comparison of Polyclonal Antisera in the Detection of Tomato Spotted Wilt Virus Using the Double Antibody Sandwich and Cocktail ELISA

Different polyclonal antisera and enzyme-linked immunosorbent assay (ELISA) procedures have been tested for their potential to detect tomato spotted wilt virus (TSWV). The virus could efficiently be detected in high dilutions of sap from infected plants, and at low concentrations of purified virus and nucleocapsid protein preparations in the cocktail ELISA and the double antibody sandwich ELISA (DAS-ELISA). Amounts of 1 to 3 ng of virus protein still gave positive readings using purified preparations, while sap could be diluted approximately 100,000 times. Differences in the detection level were observed using nucleocapsid protein antiserum (anti-N-serum) and the antiserum against intact virus particles (anti-TSWV-serum), but both antisera showed to be powerful sera for the detection of TSWV. Using anti-N-serum, TSWV could be detected in highly diluted extracts of different hosts, and also in leaf extracts or intact tissues stored for 30 days under different conditions. These results indicate that the TSWV nucleocapsid protein remains antigenic for long periods.     

双单克隆抗体ELISA间接夹心法检测流行性出血热病毒抗原

建立了检测流行性出血热(EHF)病毒抗原的双单克隆抗体(McAb)ELISA同接夹心法,用本法和间接荧光抗体技术(IFAT)相比较,IFAT检出感染细胞内病毒抗原的高峰在感染后第8天,而ELISA检测感染上清中病毒抗原的高峰在第14天,两方法检测179份人工感染EHF病毒的乳鼠脑和肺组织标本,阳性检出率分别为72.1％和68.2％,实验结果表明,本法特异,敏感,简便,不仅可用于EHF病原学研究,也适用于流行病学调查检测大量鼠肺标本。