RFFIT和MNT检测狂犬病毒中和抗体的比较研究

目的 对RFFIT和MNT两种方法检测抗狂犬病毒中和抗体的相关性,敏感性和重复性进行比较.方法 用MNT和RFFIT两种方法来检测已免疫的人血清52份,未免疫的人血清40份.结果 显示这两种方法检测已免疫血清的一致性是88.5%,其相关性是r=0.886,MNT的敏感性和特异性分别是84.9%,100%,RFFIT的灵敏度和特异性分别是100%,89.0%.MNT和RFFIT重复检测一个样品的变异系数(CV)分别是62.3%、13.3%.结论 RFFIT与MNT的相关性较好,RFFIT的灵敏度和重复性均比MNT好,因此,RFFIT在大多数需要检测RV中和抗体的情况下可替代MNT.     

RFFIT和MNT检测抗狂犬病毒中和抗体的比较研究

目的对RFFIT和MNT两种方法检测抗狂犬病毒中和抗体的相关性,敏感性和重复性进行比较。方法用MNT和RFFIT两种方法来检测已免疫的人血清52份,未免疫的人血清40份。结果显示这两种方法检测已免疫血清的一致性是88.5%,其相关性是r=0.886,MNT的敏感性和特异性分别是84.9%,100%;RFFIT的灵敏度和特异性分别是100%,89.0%。MNT和RFFIT重复检测一个样品的变异系数(CV)分别是62.3%、13.3%。结论RFFIT与MNT的相关性较好,RFFIT的灵敏度和重复性均比MNT好,因此,RFFIT在大多数需要检测RV中和抗体的情况下可替代MNT。     

建立双重荧光定量PCR方法检测蜱传疏螺旋体

目的 建立双重荧光定量PCR方法,同时检测Borrelia burgdorferi和Borrelia miyamotoi。方法 分别利用recA基因、glpQ基因合成引物探针,并优化反应条件。在单重实验的基础上建立双重荧光定量PCR方法,对该方法灵敏度和特异度进行检测,并探索其他因素对反应结果的影响。使用模拟样本和实际样本评价新方法的检测效用。结果 双重荧光定量PCR方法具有良好的灵敏度和特异度,与单重实验方法比较,检测Ct值无明显差异(P>0.05),病原浓度与检测Ct值具有良好的线性相关关系。两种病原浓度差异和其他病原的存在对检测结果无明显影响。使用该方法检测模拟蜱样本和实际蜱样本具有良好的实用性。结论 本实验建立的多重荧光定量PCR方法能快速便捷的检测两种疏螺旋体,为蜱样本病原鉴定提供了有价值的检测工具。     

狂犬病毒胶体金检测试纸的制备及初步应用

目的利用胶体金免疫层析技术建立一种快速、简便、特异的狂犬病毒检测方法。方法采用柠檬酸三钠还原法制备胶体金标记鼠抗狂犬病毒单克隆抗体,包被于结合释放垫处,分别利用纯化的兔抗狂犬病毒多克隆抗体和羊抗鼠IgG包被检测线及质控线,组装胶体金检测试纸。结果制备的狂犬病毒胶体金试纸对15个接种狂犬病毒乳鼠脑组织的检测均为阳性,与FAT结果一致;对犬瘟热病毒、犬细小病毒及犬冠状病毒细胞培养液及9个狂犬病毒阴性乳鼠脑处理液的检测均为阴性。结论用制备的狂犬病毒胶体金试纸检测狂犬病毒快速、简便、特异,具有良好的临床应用前景。     

免疫酶组织化学染色法检测脑组织中的狂犬病毒抗原

荧光抗体试验(FA)直接检测脑组织、唾液腺或皮肤组织中的狂犬病毒抗原,可以确诊狂犬病。但FA法需要昂贵的荧光显微镜和一定的观察经验,难于普遍摧广应用。本文参照WHO推荐的实验程序,建立了检测狂犬病毒抗原的免疫酶组织化学染色法,检查了104例脑组织标本,并同FA法讲行了比较,取得了满意的结果,现报告如下。 材料和方法 一、标本 狂犬病毒CVS-7株(卫生部成都生物制品研究所提供)感染发病死亡的小鼠41只,狂犬     

狂犬病病毒荧光定量RT—PCR检测方法的建立与初步应用

目的建立狂犬病病毒快速检测法。方法根据狂犬病病毒核蛋白基因的保守序列分别设计两对引物及其相应的TaqMan探针。构建pMD-N重组质粒,建立荧光定量RT-PCR绝对定量标准品,并对反应体系进行优化,建立绝对定量的标准曲线。利用10倍稀释法检验方法的灵敏度并与普通RT-PCR法进行比较;作重复性和特异性检验后进行临床标本的检测。结果构建了绝对定量的参照质粒,标准曲线相关系数为0.998。狂犬病病毒荧光定量RT-PCR检测反应的灵敏度为10个TCID_(50);5种非狂犬病病原体检测均为阴性。结论建立的狂犬病病毒的荧光定量RT- PCR检测方法快速、特异性强、灵敏度高、稳定性好,可以应用于临床样品的检测。     

贝类奥尔森派琴虫实时荧光定量PCR检测方法的建立及初步应用

目的建立一种快速、灵敏、特异的用于检测贝类奥尔森派琴虫的实时荧光定量PCR方法。方法根据基因库中奥尔森派琴虫的基因保守序列,设计合成1对引物和1条TaqMan探针,建立荧光定量PCR方法,对采自广西沿海的49份贻贝标本进行检测,并与常规PCR比较。结果建立的荧光定量PCR方法灵敏度可达20个拷贝,比常规PCR灵敏度高100倍。49份贻贝标本的阳性率为16.3%,检测的奥尔森派琴虫基因组DNA含量为2.38×10^6～9.21×10^2拷贝/μl。结论建立的荧光定量PCR方法可以用于贝类奥尔森派琴虫感染的快速检测。     

空肠弯曲菌实时荧光定量PCR检测方法的研究

目的建立一种快速廉价的检测空肠弯曲菌的实时荧光定量PCR方法。方法根据空肠弯曲菌flaA、gyrA基因设计引物,建立空肠弯曲菌实时荧光定量PCR检测方法。结果以flaA基因引物对空肠弯曲菌DNA进行实时荧光定量PCR,扩增曲线呈S形指数增长,大肠埃希菌等对照菌扩增阴性。方法的灵敏度为10CFU/ml菌悬液。结论建立的空肠弯曲菌荧光定量PCR检测方法具有快速、简便、准确等特点,具有一定的使用价值。     

实时荧光定量PCR在手足口病肠道病毒快速检测中的应用

目的建立快速检测手足口病肠道病毒的实时荧光定量PCR法,并作出应用分析。方法采用卫生部《手足口病预防控制指南》(2008年版)推荐的RT-PCR法,对丽水市2008年4～5月间发生的172例临床诊断手足口病患者的324份标本进行人肠道病毒、柯萨奇病毒A组16型和肠道病毒EV71型特异性核酸的检测,同时采用实时荧光定量PCR法进行平行检测,比较两者的实验结果,分析实时荧光定量PCR方法的特异性和灵敏度。结果实时荧光定量PCR检测324份标本,肠道病毒通用核酸阳性70份,柯萨奇A16核酸阳性22份,肠道EV71病毒核酸阳性15份,与RT-PCR结果一致,符合率100%。结论实时荧光定量PCR法具有特异性强、灵敏度高等优点,是一种理想的手足口病肠道病毒的检测方法。     

牛结核病荧光定量PCR快速检测方法的建立及初步应用

目的建立牛结核病荧光定量PCR快速检测方法。方法根据分枝杆菌的插入序列IS1081设计引物和探针,优化反应条件,建立标准曲线,进行特异性、敏感性、重复性实验,并用所建立的方法对临床样品进行检测。结果该方法的敏感性达到了10个拷贝,且特异性高,重复性好。对30份PPD试验和巢式PCR检测都为阳性的临床样本进行荧光定量PCR检测的结果全部为阳性;而对PPD检测阳性,巢式PCR检测为阴性的15份临床样本进行检测时,2份为阳性;在对2份PPD检测阴性而巢式PCR检测阳性的临床样本的检测结果也为阳性。结论成功建立了牛结核病荧光定量PCR快速检测方法,对临床样品的快速检测和牛结核病的早期诊断具有重要意义。     

一例暴露因素不明的狂犬病病例的分子流行病学溯源

目的 对一例暴露因素不明的人狂犬病病例进行实验室确诊,通过分子流行病学分析探索其可能的感染来源。方法 通过对存活疑似人狂犬病病例的唾液、脑脊液、血清标本采用直接免疫荧光试验、反转录-聚合酶链式反应和快速荧光灶抑制试验进行实验室确诊;通过时空进化分析探索该病例可能的感染来源。结果 病例唾液标本直接免疫荧光试验结果呈阳性,病例血清标本经快速荧光灶抑制试验检测抗狂犬病病毒中和抗体呈阳性。病例唾液标本经反转录-聚合酶链式反应获得狂犬病病毒核蛋白基因预期扩增片段,序列测定进一步证实为狂犬病病毒。该狂犬病病毒株与2011年安徽省一株犬源狂犬病病毒株核苷酸序列同源性最高,为98.4%。结论 该疑似狂犬病病例可确诊为狂犬病病例。其发病源于至少2011年以来的某次不自觉的狂犬病病毒感染。     

The absence of anti-rabies antibody in the sera of feral raccoons (Procyon lotor) captured in Hokkaido, Japan

Feral raccoons captured in Hokkaido, Japan were examined for the presence of rabies virus neutralizing antibody (VNA). Between 2000 to 2002, 741 serum samples were collected and then subjected to VNA titer determination by rapid fluorescent focus inhibition test (RFFIT). Sera showing RFFIT titers of > or =1:25 have been considered as positive according to previous reports. VNA was detected in none of serum samples from the feral raccoons. The present study provides valuable background information for rabies prevention in Japan. The potential risk of raccoon-derived rabies in the wilderness has been of concern because of the increasing number of feral raccoons originally introduced from rabies-endemic countries. The continuous monitoring of sick and/or dead wild raccoons would help to prevent an epidemic spread of raccoon rabies.     

Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans.

OBJECTIVES: Currently three tests are approved for the estimation of neutralizing antibodies after rabies vaccination: the mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT), and the fluorescent antibody virus neutralization (FAVN) test. Performance of these tests requires a lot of expertise and is generally carried out in reference laboratories and, hence, they are not available to many people. The aim of the present study was to develop and evaluate a competitive ELISA (C-ELISA) for estimation of neutralizing antibodies in order to make this testing more widely available. METHODS: The C-ELISA was designed based on competition between a murine neutralizing monoclonal antibody (Mab) and the antibodies in serum of vaccinated people. The test was initially standardized using known negative and known positive serum samples for determining the optimal dilution of the Mab as well as the cut-off value (%) for ascertaining the level of inhibition. Nine hundred and ninety serum samples were tested from 250 people who had been administered purified chick embryo cell vaccine (PCECV). Serum samples were collected on days 0, 14, 30 and 90 post-vaccination, and were tested by C-ELISA. RESULTS: All the serum samples that were positive by RFFIT were also positive by C-ELISA. The titers obtained with C-ELISA were marginally higher than the RFFIT titers, but a significant correlation was noted between the two tests (r=0.897). None of the negative controls were detected to be positive for rabies antibodies by either of these tests. Therefore the C-ELISA was found to be 100% specific and sensitive in comparison to RFFIT. Further, the initial rise and fall of antibody titers on different days post-vaccination was comparable for both tests. CONCLUSIONS: The C-ELISA described herein can be used to quantify rabies neutralizing antibody levels after vaccination. This test is simple and can be conveniently used under field conditions for monitoring seroconversion after post-exposure rabies vaccination. Moreover it does not require handling of infectious virus by the end user.     

Evaluation of Rapid Neutralizing Antibody Detection Test against Rabies Virus in Human Sera

Rapid and easy determination of protective neutralization antibody (NAb) against rabies in the field is very important for an early and effective response to rabies in both animal and human health sectors. The rapid neutralizing antibody detection test (RAPINA), first developed in 2009 and then improved in 2012, is a quick test allowing detection of 0.5 IU/ml antibodies in human and animal sera or plasma. This study aimed to assess the RAPINA test by comparison with rapid focus fluorescence inhibition test (RFFIT), using 214 sera of vaccinated and unvaccinated professional dog butchers, laboratory workers and rabies patients in Vietnam. The sensitivity, specificity, false negative rate, false positive rate and concordance of the RAPINA test as compared to RFFIT were 100%, 98.34%, 0%, 1.66% and 98.6%, respectively. The positive predictive value and negative predictive value were 91.7% and 100%, respectively when RAPINA test was used. With its remarkable sensitivity, specificity and easy implementation, RAPINA test can be used for rapid determination of NAb in the field.     

狂犬病疫苗接种人群中狂犬病病毒中和抗体水平分析

目的 通过分析狂犬病疫苗接种人群中狂犬病病毒RABV中和抗体特征,为狂犬病防控提供依据。方法 收集2019-2020年杭州市职业病防治院犬伤后暴露预防接种门诊抗体检测者信息,经筛选后确定157例RABV中和抗体检测者作为研究对象,采用快速荧光灶抑制试验检测狂犬病毒中和抗体,利用横断面研究方法对狂犬病毒中和抗体水平进行人群特征分析。结果 研究对象中,初种者98人,复种者59人,复种者中2-1-1程序比5针法产生的抗体水平更高,其余特征均无统计学差异(P>0.05)。除1例初种者中和抗体效价低于0.5 IU/mL,其余检测者中和抗体效价均高于0.5 IU/mL。狂犬病毒中和抗体水平时间趋势分析显示,狂犬病毒中和抗体水平与时间呈负相关,回归方程为(F=4.974,P=0.031),标准化回归系数为-0.322(t=-2.230,P=0.031)。结论 复种者狂犬病毒中和抗体水平与免疫程序有关,2-1-1程序对人体再次免疫应答的效果优于5针法。接种疫苗2年后部分个体出现失保护状态,再次暴露有必要全程接种狂犬病疫苗。     

Use of latex agglutination test to determine rabies antibodies in production of rabies antisera in horses

A therapeutic anti-rabies immunoglobulin for human use has been produced mainly in horses. The presently available seroneutralization test, the rapid fluorescent focus inhibition test (RFFIT), is laborious and rather difficult to carry out in horse farms. This study was undertaken to develop a simple latex agglutination test (LAT) for determining rabies antibodies in horse sera. LAT was validated by testing a total of 468 horse serum samples characterized by RFFIT. Of these, 253 of 260 samples with antibody titers of less than 100 IU/ml had agglutination score of 1+, whereas 174 of 208 samples with antibody titers equal to or greater than 100 IU/ml had agglutination scores of 2-4+. Results of LAT correlated with those of RFFIT (r = 0.87, p < 0.0001). LAT has the advantages of being rapid, simple to perform, easy to interpret, and applicable as an on-site testing tool for the estimation of rabies antibodies in horses.     

A Comparative Evaluation of the Estimation of Rabies Virus Antibodies among Free-Roaming,Vaccinated Dogs in Bengaluru,India

Vaccination is the practical solution for the prevention of rabies in dogs. Assessment of the immunogenicity of vaccination includes estimation of specific rabies virus neutralizing antibodies (VNA) in the target species. We undertook a study to estimate the levels of VNA in free-roaming dogs with a history of rabies vaccination in Bengaluru city, India. We compared the rapid fluorescent focus inhibition test (RFFIT) and an in-house quantitative indirect ELISA (iELISA). The study area comprised the jurisdiction of Bruhat Bengaluru Mahanagara Palike (BBMP), the Bengaluru civic body. The BBMP, along with several non-government organizations (NGO), were conducting a trap- neuter- vaccinate- release program for the prevention of dog rabies. Serum samples were collected from 250 free-roaming dogs from representative regions of BBMP, of which 125 had a VNA titre of 0.5 IU or more by the RFFIT. Furthermore, 126 dogs showed percent positivity values (PP values) more than the cut off PP value of 57.1 by the iELISA, accounting for 50.4% of satisfactory post-vaccinal serum conversion. The sensitivity and specificity of the iELISA was 94.4% and 95.2%, respectively. Based on these data, a quantitative iELISA may be a complementary tool for sero-monitoring immune responses of free-ranging animals after rabies vaccination.     

冀、京、吉犬群犬腺病毒中和抗体调查

目的为表达狂犬病病毒糖蛋白的犬2型腺病毒（CAV-2）重组狂犬病口服疫苗的投放工作提供流行病学依据。方法通过病毒中和试验对随机采自河北省,北京市,吉林省等地的387份犬血清进行犬腺病毒中和抗体检测,以确定犬群腺病毒感染率。结果河北、吉林农村流浪犬或家养犬腺病毒中和抗体阳性率较低,阳性率为16％（30／192）,而北京市犬腺病毒中和抗体阳性率高达69％（135／195）,说明城乡犬只犬腺病毒中和抗体存在明显差异。结论农村流浪犬或家养犬腺病毒中和抗体阳性率较低,适用于CAV-2及其重组疫苗首次免疫、尤其是口服免疫。     

Anti-rabies virus IgM in serum and cerebrospinal fluid from rabid dogs

An anti-rabies IgM antibody capture radio immunoassay was used to test serum and cerebrospinal fluid from 37 dogs held in quarantine for suspicion of rabies. Rabies was confirmed in dogs that died by mouse inoculation and subsequent examination of mouse brains by fluorescent antibody technique to detect rabies antigen. The mean counts per minute (CPM) of iodinated anti-rabies gamma globulin coupled IgM rabies antibody in CSF and serum from rabid dogs were significantly higher than in CSF and serum from non-rabid dogs. Mean CPM from rabid dogs was greater in CSF than in sera, in contrast with non-rabid dogs, from which mean cpm was higher in sera than CSF, suggesting that antibody may have been synthesized in the CSF. To evaluate this test further, a dog was infected by rabies virus, and serial serum and CSF specimens were collected until the time of death. IgM anti-rabies antibody developed in the CSF and serum 29 days following infection, and rose just before the dog died of rabies on day 34. The rabies MAC RIA is potentially useful as a diagnostic method in quarantined dogs with rabies-like illness. Perhaps more importantly, it may be applied to better understand the immunopathogenicity of rabies.