口蹄疫病毒TaqMan荧光定量PCR检测方法的建立

为建立检测口蹄疫病毒(FMDV)的方法,本研究根据GenBank中FMDV的2B基因序列,设计合成一对引物和一条TaqMan探针,将2B基因克隆到pBlueScriptSK(-)载体中,利用T7体外转录试剂盒制备标准品,通过优化反应条件,建立了TaqMan荧光定量PCR检测方法.结果表明,该检测方法的敏感性达到102拷贝/μL;与其它主要相关病毒均不发生交叉反应,批内和批间试验重复性的变异系数(CV)均小于3％.本研究建立的FMDV TaqMan荧光定量PCR方法对FMDV的快速检测具有重要意义.     

A rapid, quantitative assay for titration of bovine virus diarrhoea-mucosal disease virus

An end point dilution microtitration assay is described that can be used for the titration of both cytopathic and non-cytopathic isolates of bovine virus diarrhoea-mucosal disease virus. Indirect immunofluorescence is used to detect infected MDBK cells in the wells of Terasaki plates. The virus titre is derived from the number of uninfected wells, using the Poisson distribution. The assay is simple, fast and economical. Titres of cytopathic virus determined by the microtitration assay and standard plaque assay are equivalent.     

A rapid virus neutralization assay for Newcastle disease virus with the swine testicular continuous cell line.

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.     

鸡新城疫病毒荧光RT-PCR与病毒分离方法的比较

2006年冬春季,在广州附近地区的一些竹丝鸡场、三黄鸡场、珠珍鸡场的35~50日龄鸡群相继暴发了以高死亡率、伴有呼吸道和神经症状的疫情。病鸡张口伸颈呼吸,尖叫、咳嗽,脚软,剖检病变以支气管堵塞为特征,偶见肺充血、出血、盲肠栓子、腺胃出血,病程10 d左右,死亡率20%~40%,愈后鸡群出现较多有神经症状鸡及咳嗽的鸡。我们到6个发病鸡场(A、C、D、F、G、H),现场调查流行病学、临床症状,并收集待检样本材料,同时进行新城疫病毒荧光RT-PCR检测与常规病毒分离鉴定诊断,比较两1材料与方法1.1待检病例样本包括血清样本Aa(A场发病4日龄鸡群)、C…     

A rapid enzyme-linked immunosorbent assay for the detection of foot-and-mouth disease virus in epithelial tissues

A rapid double sandwich enzyme-linked immunosorbent assay (ELISA) has been used for the identification and type differentiation of foot-and-mouth disease (FMD) viruses in epithelial tissue samples submitted for diagnosis from the field. No difficulty was experienced in the direct typing of freshly harvested epithelium from recently ruptured vesicles by the complement fixation (CF) test or ELISA. The ELISA was more sensitive and specific, but proved no more efficient than the traditional CF test in the direct typing of samples of poorer quality from many countries overseas where communications are often difficult. However, when both tests were used concurrently, FMD virus typings were confirmed in 27 more samples. Some possible reasons for the failure of ELISA to detect virus in certain cases are discussed.     

Real-time RT-PCR和RT-PCR方法快速检测犬瘟热病毒

由犬瘟热病毒(CDV)引起的犬瘟热(CD),是犬的一种高度接触性、急性传染病[1].CDV感染范围广泛,自然宿主包括犬科动物(犬、狼、豺、狐等),鼬科动物(貂、臭鼬、黄鼠狼等),浣熊科动物(浣熊、小熊猫、大熊猫等),还可感染灵长类动物(如日本猕猴等),甚至有CDV感染人的病例.除病毒分离、病理包涵体检查、血清学检测、核酸探针、原位杂交等方法外,RT-PCR方法是目前检测CDV最常用的方法[1-3].     

梅迪-维斯纳病毒实时荧光定量PCR检测方法的建立

为建立检测梅迪-维斯纳病毒(MVV)的TaqMan实时荧光定量PCR方法,本研究根据MVV核苷酸保守序列设计引物和探针。以梯度稀释的含有MVV目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示:5.0×105～5.0×101拷贝范围内定量PCR均有S型扩增曲线,检测灵敏度为50拷贝。对羊的其他病毒核酸均无扩增反应。本研究建立的实时定量PCR方法,灵敏度高,特异性好,在MVV的快速检测中具有良好的应用前景。     

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。     

Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines

To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.     

鹅副粘病毒实时荧光定量RT-PCR检测方法的建立

根据CenBank所载鹅副粘病毒(CPMV)的F基因保守区序列设计一对特异性引物,应用RT-PCR方法扩增F基因片段,克隆、测序后,采取Taqman探针法建立了检测鹅副粘病毒的荧光RT-PCR方法.结果表明,该方法特异性强,敏感性高,可以用于鹅副粘病毒的检测.     

塞内卡谷病毒和口蹄疫病毒双重荧光RT-PCR检测方法的建立

为建立塞内卡谷病毒(SVV)和口蹄疫病毒(FMDV)的快速鉴别检测方法,本试验根据SVV 3D基因序列和FMDV 3D基因序列,分别设计探针和引物,建立了可同时检测SVV和FMDV的双重荧光RT-PCR法。结果表明,该方法特异性强,能准确检测出SVV核酸和FMDV核酸,与水泡性口炎病毒(VSV-IND和VSV-NJ)、猪水泡病病毒(SVDV)、猪繁殖与呼吸综合征病毒(PRRSV)以及猪瘟病毒(CSFV)等病原核酸无交叉反应;灵敏度高,本试验中最低可检测到SVV和FMDV核酸质量浓度分别为1.35×10~(-5),1.68×10~(-5) mg/L。重复性好,Ct值变异系数小于4%。利用所建立方法对116份已知的临床样品进行检测,结果与参比方法一致。本试验建立的方法可用于SVV和FMDV的鉴别检测,为塞内卡病毒病和口蹄疫的诊断提供依据。     

禽流感病毒一步法RT-PCR检测方法的建立

为了能快速准确地对禽流感进行病原学诊断，根据流感病毒的M基因序列，在保守区内用Olig04．0软件设计了1对引物，建立了一步法RT—PCR诊断方法，其目的片段大小为229bp。通过对不同稀释倍数的H5亚型禽流感病毒尿囊液和棉拭子浸出液进行检测，结果显示，病毒尿囊液的最低检出稀释度为1：10^4；阳性棉拭子的最低检出稀释度为1：2^3。用病毒分离法和一步法RT—PCR同时检测人工感染鸡不同脏器、口咽及泄殖腔棉拭子样品，二者符合率为100％，但前者的检测灵敏度比后者高10～100倍。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原，所有禽流感病毒均有229bp的目的条带出现，而其他14种禽病病原均无目的条带出现，表明该方法的特异性好。     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine virus diarrhea virus in sera from border disease virus-infected sheep.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.     

牛呼吸道合胞体病毒RT-PCR检测方法的建立及初步应用

本研究旨在评估日粮补充以枯草芽孢杆菌、乳酸菌和酵母菌组成的复合益生菌对育肥猪生长性能、小肠绒毛形态及肝脏脂代谢相关指标的影响。试验将208头体重接近的18周龄三元育肥猪随机分为2组,每组4个重复,每个重复26头。对照组育肥猪饲喂基础日粮,处理组育肥猪饲喂基础日粮+1.5 g/kg由乳酸菌、枯草芽孢杆菌和酵母菌组成的复合益生菌,饲养试验时间为8周。结果：处理组试验28 d育肥猪体重、1～28 d和1～56 d平均日增重较对照组显著提高2.28%、10.33%和4.56%（P＜0.05）,但1～28 d和1～56 d料重比显著降低11.15%和5.72%（P＜0.05）。处理组空肠绒毛高度、绒毛高度与隐窝深度比值较对照组分别显著提高5.45%和17.52%（P＜0.05）,而隐窝深度显著降低10.27%（P＜0.05）,对照组与处理组育肥猪血清低密度脂蛋白和高密度脂蛋白浓度及肝脏胆固醇、高密度脂蛋白和低密度脂蛋白浓度无显著差异（P＞0.05）,但处理组育肥猪血清甘油三酯和胆固醇浓度及肝脏甘油三酯浓度显著低于对照组（P＜0.05）。结论：在本研究条件下,日粮添加1.5 g/kg以枯草芽孢杆菌、乳酸菌和酵母菌组成的复合益生菌可通过改善空肠绒毛形态,提高育肥猪生长前期和全期的平均日增重和饲料效率,降低肝脏和血清胆固醇浓度。 [关键词]益生菌|育肥猪|生长性能|绒毛形态|脂代谢     

Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus

A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs.     

A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID₅₀. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.