中欧预防控制口蹄疫2011年培训3.ppt

Diagnostic Tests for FMD and New Developments in Diagnosis of FMD 口蹄疫诊断方法与最新进展Training to Improve Control and Prevention of Foot and Mouth Disease(FMD),旨在提高口蹄疫预防与控制行动的培训旨在提高口蹄疫预防与控制行动的培训Lanzhou,China,8-12 August 2011Chris MorrissyCSIRO Livestock Industries,Australian Animal Health Laboratory(AAHL),Geelong,Australia.Outline（概要）Disease Diagnosis&Control（疫病诊断与控制）Detection of FMD Virus/Viral Antigen（病毒抗原检测）FMD Serology（口蹄疫血清学检测）Quality Control（质量控制）New Developments in Diagnosis（诊断技术的新进展）Summary（总结）Disease Diagnosis and Control疫病诊断与控制疫病诊断与控制Animal Health Network(Outbreak)动物健康工作网络（暴发疫情）动物健康工作网络（暴发疫情）Field Veterinarians（田间兽医）（田间兽医）clinical diagnosis（临床诊断）（临床诊断）collect information/history of disease,vaccination&recent animal movement（收集信息收集信息/病史，免疫与最近病史，免疫与最近动物的移动情况）动物的移动情况）collect samples for laboratory（采集实验室诊断样品）采集实验室诊断样品）implement disease control（采取控制措施）（采取控制措施）quarantine&movement control（检疫与移动控制）（检疫与移动控制）Disinfection（消毒）（消毒）Disease Diagnosis and ControlAnimal Health Network(Outbreak)Laboratory（实验室）confirm clinical diagnosis（临床诊断的确诊）Sero-typying ELISA（血清定型ELISA）PCR&sequencing(PCR与序列测定)virus isolation（病毒分离）molecular epidemiology&characterisation（分子流行病学与特征分析）vaccine matching 疫苗配比 send samples to RRL&WRL （向RRL与WRL发送样本）share information 信息共享Diagnosis of FMD（诊断）（诊断）Clinical Diagnosis（临床诊断）（临床诊断）initial diagnosis based on clinical signs by a trained or experienced investigator 由专业人员根据临床症状作出的初步诊断）important to send history of disease outbreak and description of lesions（重点是对病史与病灶的描述）should also involve an epidemiological investigation to connect to other outbreaks 同时联系其他疫情开展流行病学调查 samples should be collected to confirm the serotype of FMD in the outbreak especially in new outbreak areas对新发地区尤其应当采集样品以确定FMD血清型Diagnosis of FMDLaboratory Diagnosis（实验室诊断）the role of the laboratory is generally to confirm the clinical diagnosis 实验室诊断是为了进一步确认临床诊断实验室诊断是为了进一步确认临床诊断it may also confirm a suspicion of disease activity(PCR or serology)也可以用于对也可以用于对PCRPCR与血清学阳性疑似疫情进行确诊与血清学阳性疑似疫情进行确诊and to provide additional information about the agent involved（进一步了解病原因子的其他信息）进一步了解病原因子的其他信息）serotype of FMD（血清型）（血清型）molecular epidemiology（分子流行病学）（分子流行病学）vaccine matching（疫苗配比）（疫苗配比）Send large tissue samples:1 2 grams送检送检1-21-2克的发病组织样品克的发病组织样品Disease Diagnosis and ControlAnimal Health Network(surveillance)Animal Health Network(surveillance)（疫情监测）（疫情监测）（疫情监测）（疫情监测）Field Veterinarians 田间兽医田间兽医 serosurveillance for disease prevalence 疫病发生状况的血清学监测疫病发生状况的血清学监测 serosurveillance post vaccination免疫后的血清学监测免疫后的血清学监测 collect information/history of disease&vaccination&vaccine used 收集信息、病史与免疫与疫苗使用情况收集信息、病史与免疫与疫苗使用情况 investigate possible vaccine failure 调查疫苗免疫失败的可能性调查疫苗免疫失败的可能性 collect samples for laboratory 采集样本用于实验室诊断采集样本用于实验室诊断Disease Diagnosis and ControlAnimal Health Network(surveillance)动物健康工作网络（监测）动物健康工作网络（监测）Laboratory（实验室）（实验室）confirm disease prevalence 确认疫病流行状况确认疫病流行状况 confirm post vaccination antibody 确认免疫后抗体产生情况确认免疫后抗体产生情况 virus characterisation(carrier animals)病毒特征分析（带毒动物）病毒特征分析（带毒动物）Disease Diagnosis（诊断）（诊断）Panel of Tests（检测方法）（检测方法）Detection of Virus/Viral Antigen 检测病毒抗原检测病毒抗原 Detection of Antibody 检测抗体检测抗体 Production of reagents 试剂生产试剂生产 Rapid,Sensitive and Specific 快速，敏感和特快速，敏感和特异异 Choose Test fit for purpose 根据目的选择方法根据目的选择方法Diagnostics for Detection of Virus/Viral Antigen（病原（病原检测方法）方法）Virus Isolation 病毒分离 Molecular Techniques 分子检测技术 Electron Microscopy 电子显微镜 Antigen Capture ELISA 抗原捕获ELISA Quality Assurance 质量保证Virus Isolation 病毒分离病毒分离 Virus isolation requires specialized facilities for cell culture&handling of virus 要求具有专门用于操作病毒与细胞培养的设施要求具有专门用于操作病毒与细胞培养的设施 Cell culture is expensive to maintain 细胞培养是昂贵的细胞培养是昂贵的 Reference laboratories need to grow virus to allow further characterization 参考实验室需要培养病毒用于进一步研究参考实验室需要培养病毒用于进一步研究 FMD virus isolates important in comparing current field isolates against vaccines being used.(R-Value/vaccine matching)病毒分离是进行流行毒与疫苗毒抗原比较的基础病毒分离是进行流行毒与疫苗毒抗原比较的基础（R R值值/疫苗配比）疫苗配比）FMD virus isolates used for molecular epidemiology 用于分子流行病学研究用于分子流行病学研究Molecular Technologies 分子分子检测技技术 PCRPCR Conventional PCR/RT-PCR 传统PCR group&specific primers(FMD serotyping)群与型特异性引物群与型特异性引物 （鉴定血清型）（鉴定血清型）Real-time PCR 实时荧光实时荧光PCRPCR group primers&probe:FMD（群引物与探针）（群引物与探针）Pirbright&Plum Island test specific primers&probe for sero-typing are needed 需要血清型特异性的引物与探针需要血清型特异性的引物与探针 Sequencing PCR productSequencing PCR product （PCRPCRPCRPCR产物测序）产物测序）产物测序）产物测序）confirm PCR product is from FMDV 为为FMDVFMDV病毒基因病毒基因 sero-typing FMDV 血清型鉴定血清型鉴定 molecular epidemiology 分子流行病学分析分子流行病学分析Molecular Technologies 分子分子检测技技术Technology is used in reference laboratories to Technology is used in reference laboratories to undertake genetic studies to link viruses into undertake genetic studies to link viruses into familiesfamilies（参考实验室利用该方法主要是进行遗传关系研（参考实验室利用该方法主要是进行遗传关系研（参考实验室利用该方法主要是进行遗传关系研（参考实验室利用该方法主要是进行遗传关系研究，以确定病毒类型）究，以确定病毒类型）究，以确定病毒类型）究，以确定病毒类型）sequencing&sequence analysis is best carried out at a Reference laboratory（测序与序列分析最好是在（测序与序列分析最好是在参考实验室进行）参考实验室进行）Epidemiological studies-molecular epidemiologyEpidemiological studies-molecular epidemiology 流行病学研究流行病学研究分子流行病学分子流行病学compare field strain to vaccine strain（比较田间毒株（比较田间毒株与疫苗毒株）与疫苗毒株）virus source and evolution（病毒来源与进化）（病毒来源与进化）FMD Antigen Capture ELISA（抗原捕获（抗原捕获ELISAELISA）Test of choose for countries endemic with FMD 该方法用于该方法用于FMDFMD流行国家流行国家 Pirbright reagents generally used(expensive)一般用英国参考实验室的试剂（较昂贵）一般用英国参考实验室的试剂（较昂贵）Allow sero-typing of FMDV(need for PCR)（鉴定血清型）（鉴定血清型）Routinely test for serotypes O,A&Asia 1（通常测定（通常测定O O、A A、Asia1Asia1）Result in 3-4 hours（3 34 4小时出结果）小时出结果）Sero-typing important for control by vaccination 鉴定血清型是疫苗免疫的重要依据鉴定血清型是疫苗免疫的重要依据 Lesions older than 4 to 5 days reduced antigen 发病发病4 45 5天的陈旧病料含毒量低天的陈旧病料含毒量低 Samples unsuitable for virus isolation(VI)&PCR can be used in the ELISA（不适于进行病毒分离和不适于进行病毒分离和PCRPCR的样品可以用于的样品可以用于ELISAELISA）FMD Antigen Capture ELISA(抗原捕抗原捕获ELISA)Sero-typing ELISA(定型ELISA)Serotype specific so 7 separate tests required on suspect sample(also VSV x 2&SVD)（每个可疑样本要进行（每个可疑样本要进行7个个测试以确定血清型（也要测试测试以确定血清型（也要测试VSV与与SVD）Most countries test for circulating sero-types (O,A,Asia1)(Sat1,2,3 also in Africa)多数国家只测定流行的血清型（O,A,Asia1）（在非洲还要测南非在非洲还要测南非3个型）个型）Positive is regarded as diagnostic confirmation（有阳性结果（有阳性结果就可以作出确诊）就可以作出确诊）Electron Microscope&PCR used as well if available（电镜与PCR也可以）Confirm CPE result in cell culture with antigen detection ELISA（细胞培养出现CPE后要用抗原捕获ELISA进行确认）FMD Antigen Capture ELISA(抗原捕抗原捕获ELISA)Does not require infectious virus,but does require a good specimen from a fresh lesion(要求要求样品采自新鲜的病灶样品采自新鲜的病灶)Antigen can be destroyed by excessive heat or presence of disinfectant（高热或消毒剂都会破坏抗原）高热或消毒剂都会破坏抗原）PCR&VI combined with ELISA increases chance of a result PCR/PCR/病毒分离结合病毒分离结合ELISAELISA定型以提高确诊的机会定型以提高确诊的机会FMD Virus Isolation（病毒分离）（病毒分离）Cell Culture（细胞培养）（细胞培养）Primary bovine(calf)thyroid cell cultures considered most sensitive 小牛甲状腺细胞是分离病毒最敏感的小牛甲状腺细胞是分离病毒最敏感的 Primary pig,calf or lamb kidney cell cultures 原代肾细胞原代肾细胞 Cell lines BHK(baby hamster kidney),IBRS2(swine kidney cell line)sometimes less sensitive for field strains and PK-15(BHK,IBRS2(BHK,IBRS2与与PK-15PK-15细胞对田间毒株略为不敏感细胞对田间毒株略为不敏感)FMD virus grows quickly and cytopathic effect can usually be seen at first or second pass in primary cells;can take longer in cell lines（FMDVFMDV在原代细胞生长迅速，通常在原代细胞生长迅速，通常1 12 2代就能出现明显的代就能出现明显的CPECPE，在传代细胞系所需时间会长一些），在传代细胞系所需时间会长一些）FMD Virus Isolation（病毒分离）（病毒分离）virus isolation requires live virus but can detect smaller amounts（病毒分离需要的活病毒的量较小）（病毒分离需要的活病毒的量较小）virus isolation requires specialized facilities（要求（要求有专门设施）有专门设施）reference laboratories need to grow virus to undertake vaccine comparison studies（参考实验室需要（参考实验室需要培养病毒来进行疫苗抗原配比研究）培养病毒来进行疫苗抗原配比研究）virus used for looking at R value comparing current field isolate against vaccines being used（通过测定（通过测定R R值来比较当前流行毒与疫苗毒的抗原关系）值来比较当前流行毒与疫苗毒的抗原关系）virus isolate used for genetic studies （用分离病毒进行遗传关系研究）（用分离病毒进行遗传关系研究）Molecular Technologies:FMD（分子（分子诊断技断技术）PCR PCR Conventional PCR/RT-PCR（传统（传统PCRPCR）*Group&serotyping primers（群与型鉴定引物）（群与型鉴定引物）Real-time PCR（实时荧光（实时荧光PCRPCR）Group primers&probe(*confirms FMD but not serotype)（群特异性引物与探针，不能定血清型）（群特异性引物与探针，不能定血清型）Pirbright&Plum Island test Sequencing PCR product Sequencing PCR product （PCRPCRPCRPCR产生测序）产生测序）产生测序）产生测序）confirm PCR product is from FMDV（确定基因来自FMDV）sero-typing(确定血清型)geno-typing:molecular epidemiology 基因型：分子流行病学 information on vaccine to be used（以上信息指导疫苗免疫）Molecular Technologies:FMD（分子诊断技术）（分子诊断技术）Detection of virus genetic information Detection of virus genetic information（检测病毒的遗传信息）（检测病毒的遗传信息）（检测病毒的遗传信息）（检测病毒的遗传信息）Latest technology that detects the nucleic acid that makes up the genetic code（最新技术是检测包含遗传密码的核酸序列）（最新技术是检测包含遗传密码的核酸序列）PCR used for primary diagnosis where sample quality allows（如果样品质量允许可以用（如果样品质量允许可以用PCR作初步诊断）作初步诊断）PCR for confirmation diagnosis（采用（采用PCR进行确诊）进行确诊）Expensive and not necessary where FMD is an endemic problem(serotype important)分子诊断比较昂贵，在分子诊断比较昂贵，在FMDFMD流行区不是很必要流行区不是很必要 （血清定型诊断更重要）（血清定型诊断更重要）Molecular Technologies:FMD（分子诊断技术）（分子诊断技术）Technology is used in reference laboratories to undertake genetic studies to link viruses into families 参考实验室利用该技术进行遗传关系研究参考实验室利用该技术进行遗传关系研究Epidemiological studies:molecular epidemiology 流行病学研究：分子流行病学field strain to vaccine strain（流行毒与疫苗毒关系）（流行毒与疫苗毒关系）virus source and evolution（疫源与进化分析）（疫源与进化分析）FMDV Serology（血清学（血清学诊断）断）C-ELISA(structural)(OIE)测定结构蛋白抗体的竞争ELISA（OIE）serotype specific（血清型特异性）（血清型特异性）7 ELISAs(minimum)（至少（至少7 7种种ELISAELISA试剂）试剂）Liquid-phase(LP)blocking ELISA（液相阻断（液相阻断ELISAELISA）Standard test used to determine protection from vaccination 是测定疫苗免疫保护水平的标准方法是测定疫苗免疫保护水平的标准方法 Non-structural protein antibody tests 非结构蛋白抗检测方法非结构蛋白抗检测方法 Standard test to determine antibody from infection 检测感染产生抗体的标准方法检测感染产生抗体的标准方法 Virus Neutralisation test(OIE)病毒中和试验（病毒中和试验（OIEOIE）Virus Neutralisation test 病毒中和试验病毒中和试验BHK-21,IB-RS-2&lamb kidney cells(LK)细胞系细胞系BHKBHK，IBRS2IBRS2与羔羊肾细胞（与羔羊肾细胞（LKLK）results 2 to 3 days（2 23 3天出结果）天出结果）current gold standard test 目前的金标准方法目前的金标准方法 titres of 1:32 or less are considered negative 抗体滴度小于等于抗体滴度小于等于1:321:32为阴性为阴性antibodies appear as early as 7 days,in most by 14 days post-infection抗体最早在感染后抗体最早在感染后7 7天出现，多数在感染后天出现，多数在感染后1414天出现天出现FMDV non-structural protein antibody tests非非结构蛋白抗体构蛋白抗体检测方法方法 3ABC C-ELISA（3ABC3ABC竞争竞争ELISAELISA）Prionics(Cedi)Baculo expressed NS antigen&MABs 杆状病毒表达杆状病毒表达NSNS抗原与单抗抗原与单抗AAHL Baculo expressed&chicken antibody 杆状病毒表达杆状病毒表达NSNS抗原与鸡抗体抗原与鸡抗体Indirect NS ELISA Kits（间接（间接ELISAELISA试剂盒）试剂盒）Intervet/Bommeli,UBI,Panaftosa&SvanovaRuminant&Porcine ELISAs（区分反刍兽与猪）（区分反刍兽与猪）E-coli expressed NS antigen（大肠杆菌表达（大肠杆菌表达NSNS抗原）抗原）NS peptide（NS NS 肽段）肽段）FMDV non-structural protein antibody tests非结构蛋白抗体检测方法非结构蛋白抗体检测方法VIAA(3D)AGID（VIAA琼脂扩散试验）AAHL 3D C-ELISA(3D LP-ELISA)Enzyme-linked immunoelectrotransfer blot assay(EITB)Sth Amercia 酶联免疫电转印（酶联免疫电转印（EITBEITB）,用于南美洲用于南美洲Western blotCombination of NS proteins（五种（五种NSNS蛋白）蛋白）Confirmatory Test（确认性的诊断方法）（确认性的诊断方法）AAHL FMDV 3ABC NS C-ELISA AAHL/IAEA funded project 澳大利亚动物健康实验室澳大利亚动物健康实验室/国际原子能机构资助项目国际原子能机构资助项目Competition ELISA:can be to test all species 竞争竞争ELISA:ELISA:可检测所有易感动物可检测所有易感动物Detects antibody from all serotypes 适用于所有的血清型适用于所有的血清型Detects antibody from NS proteins(3ABC)检测非结构蛋白检测非结构蛋白3ABC3ABC抗体抗体Detects antibodies from infected animals(Indicates infection&possible Carrier status)检测感染动物抗体，指示感染与带毒状况检测感染动物抗体，指示感染与带毒状况Preparedness for Disease Control 疫病防控的疫病防控的战备状状态 detailed protocols on handling outbreak 疫病发生时的详细操作程序疫病发生时的详细操作程序 trained field&laboratory staff 训练有素的野外与实验室工作人员训练有素的野外与实验室工作人员 diagnostic network from field to laboratory in place 从田间到实验室的诊断工作网络从田间到实验室的诊断工作网络 sample collection materials available to field vets to allow same day collection and transport to laboratory 田间兽医能够随时拿到样品采集的物资，以便在疫病发生的田间兽医能够随时拿到样品采集的物资，以便在疫病发生的同一天将样本采集运送到实验室同一天将样本采集运送到实验室 diagnostics established&in use inlaboratories to allow same day result 所用的实验室与诊断方法能够在同一天得出结果所用的实验室与诊断方法能够在同一天得出结果Quality Assurance(QA)Quality Assurance(QA)（质量保证）（质量保证）（质量保证）（质量保证）Ensures tests are preformed to a standard 依据某一标准开依据某一标准开展检测展检测 Paperwork/Documentation for all tests 记录所有的测试数据记录所有的测试数据 Internal Quality Control(IQC):Positive&Negative controls used in each test run 内部质量控制：每一个测试都要有阳性与阴性对照内部质量控制：每一个测试都要有阳性与阴性对照 Charting/documenting IQC controls used in each test 记录记录或填写每一个测试的内部质量控制措施或填写每一个测试的内部质量控制措施 calibration of equipment 设备的校准设备的校准Laboratory Accreditation to a StandardLaboratory Accreditation to a Standard实验室质量标准认证实验室质量标准认证实验室质量标准认证实验室质量标准认证 AAHL accredited to ISO 17025 AAHL通过了通过了ISO17025认证认证 Quality Assurance(QA)质量保证质量保证 Internal Quality Control(IQC)Internal Quality Control(IQC)内部质量控制内部质量控制内部质量控制内部质量控制 IQC controls must standardised against Reference Controls 质控必需依据标准对照进行标准化质控必需依据标准对照进行标准化 Charting IQC results(Graph&Sheet)质控结果以图质控结果以图表形式出示表形式出示 Use of Low Positive controls important to confirm sensitivity 应用弱阳性对照来确认检测的敏感性应用弱阳性对照来确认检测的敏感性 Important for test validation 质控对方法验证很重要质控对方法验证很重要 External Quality Assurance(IQA)External Quality Assurance(IQA)外部质量保证外部质量保证外部质量保证外部质量保证 Proficiency Testing(PT)熟练程度测定熟练程度测定Samples from Reference Laboratory tested in your laboratory 在你们的实验室检测来自参考实验室的样品在你们的实验室检测来自参考实验室的样品 Important for test validation IQA对于检验能力验证很重要对于检验能力验证很重要Suspect case of diseaseLOCALCLINICALOBSERVATIONLABORATORYDIAGNOSIS(Local or NRL)New Developments in Diagnosis 诊断方法的新断方法的新发展展1.Lateral-flow devices for FMDV antigen 检抗原的试纸条2.Mobile PCR 移动PCR3.Isothermal assays 等温扩增检测技术 Faster(more rapid detection)更快速 Ability to support diagnosis based on clinical signs 支持临床诊断支持临床诊断疑似病例临床观察临床观察实验室诊断实验室诊断Lateral-flow devices for FMDV Antigen detectionLateral-flow devices for FMDV Antigen detection检测检测检测检测FMDVFMDVFMDVFMDV抗原的试纸条抗原的试纸条抗原的试纸条抗原的试纸条Developed by IAH in collaboration with Brescia and Svanova 由由IAH,Brescia与与Svanova合作研发合作研发Quick and simple to perform 快速简单快速简单Pan-serotypic 适用于多个血清型适用于多个血清型During 2007:used for rapid(10 mins)confirmation of FMD in the field 20072007年：用于快速确认田间疫情（小于年：用于快速确认田间疫情（小于10min10min）Also useful in the Lab 也可用于实验室也可用于实验室LFD marketed by 由由SvanovaSvanova销售销售Progress with the Enigma Diagnostics FLProgress with the Enigma Diagnostics FL实时荧实时荧光光检测检测技技术术Enigma have licence to use TaqMan PCR technologybasis for routine assays used in most diagnostic labs(FMDV,ASFV,BTV,BVDV,CSFV etc)2009-2010:Evaluation of machines capability at IAHwet assay with MagNA PURE reagentsperformance can be equivalent to routine rRT-PCR assays used in the WRLResults in 60 minutes（60min内出结果)Future project to develop a dry-down consumable for FMDV?Nucleic acid amplification at a single temperature 等温条件下扩增 No need for fragile precision instrumentation 不需精密设备 More suitable for use in the field 适合野外条件 Very rapid,similar sensitivity to rRT-PCR 快速，与实时荧光快速，与实时荧光PCRPCR敏感性相似敏感性相似Isothermal amplification(RT-LAMP)等温扩增技术等温扩增技术Dukes et al.,2006James et al.,2010Alternative amplification technologies可选的扩增技术可选的扩增技术可选的扩增技术可选的扩增技术SimpleRapidSensitiveDisposable test?Opportunities for non-invasive samplingOpportunities for non-invasive sampling非侵入性采样非侵入性采样非侵入性采样非侵入性采样FMDV