细小病毒抗原快速检测卡临床使用效果评价

本试验以PCR方法为标准,以临床收集的213份粪便样本为检测对象,对3家商品化犬细小病毒抗原快速检测卡的临床应用效果进行了评价:与PCR方法相比较,3家产品各自的敏感性分别为71.7%,66.4%和72.2%;相对应的特异性分别为96%,97%和96%。3家检测卡彼此之间的符合率分别为96.7%、99.5%和95.8%。以PCR方法和其中两种检测卡对同一窝5只感染犬细小病毒的幼犬的临床病征与CPV检测结果进行相关性评估,结果显示,犬细小病毒抗原快速检测卡的阴性检测结果与病犬临床症状的缓解或消失时间呈正相关,而PCR方法在动物临床病征消失后仍可检出病毒。     

犬细小病毒血凝抑制抗体效价胶体金免疫层析检测方法的建立及应用

试验旨在利用胶体金免疫层析技术建立快速检测犬血清中犬细小病毒（canine parvo virus, CPV）血凝抑制（haemagglutination inhibition, HI）抗体效价的方法,用于CPV疫苗免疫效果评价。采用双抗体夹心法,以抗CPV血凝相关抗原的单克隆抗体制备CPV抗原检测试纸条;将犬血清进行不同比例系列稀释后,分别与定量CPV抗原充分反应,滴入CPV胶体金试纸条,根据试纸条检测线（test line,T线）消失时的血清最高稀释倍数判断血清中CPV抗体的HI效价;用此方法检测86份犬血清样品,并与传统血凝抑制试验方法进行分析比较。结果显示,成功制备CPV抗原检测试纸条,确定了试纸条检测犬血清CPV-HI效价的反应条件和结果判定标准。结果表明,在检测不同稀释倍数犬血清反应后的CPV抗原时,能使试纸条T线消失时的血清最高稀释倍数与HI效价具有正相关性,犬血清最高稀释倍数乘以4即为HI效价;两种方法的符合率达90.7%。本试验初步建立了胶体金试纸条检测CPV血凝抑制效价的方法,为检测CPV-HI效价提供了一种操作简单、快速的试验方法,可用于CPV疫苗免疫效果评价。     

犬细小病毒CPV-QD12株的分离鉴定与序列分析

本试验成功分离了1株犬细小病毒,采用PCR、理化特性检验、HA及HI等方法对其进行了鉴定,并对其编码区全基因序列进行了分析。取临床患出血性肠炎幼犬粪便经无菌处理后同步接种F81猫肾细胞分离病毒,盲传至第4代开始出现典型的细胞病变。该病毒可凝集猪的红细胞,血凝效价为28,可被犬细小病毒单克隆抗体特异性中和而产生血凝抑制现象;病毒效价为105.5TCID50/m L;毒株对氯仿和胰酶不敏感,且耐酸耐热。经病毒理化特性试验及犬细小病毒单克隆抗体的血凝抑制鉴定其为犬细小病毒,通过PCR检测及编码区全基因的扩增、测序和序列比较,证实所分离毒株为CPV-2a亚型,并将其命名为CPV-QD12株,与2013年分离自中国江苏省的CPV-2a型犬细小毒株CPV-JS2及2011年分离自中国广西的CPV-2a型毒株CPV-JQ686671.1的编码区核苷酸相似性为99.6%,具有较近的亲缘关系。该研究可为诊断和防控犬细小病毒病提供参考。     

犬细小病毒TZ2#株的分离与鉴定

采集疑似犬细小病毒(CPV)感染犬的粪便,采用同步培养法接种胎猫肾细胞(FK81)进行病毒分离鉴定。通过PCR检测、HA试验、PEG纯化,获得1株犬细小病毒,并命名为TZ2#株。感染的F81细胞48h后出现明显的细胞病变;在感染的FK81细胞中扩增出CPV基因的特异性片段;病毒液可凝集猪红细胞,血凝价为1∶64,其血凝性能被特异性抗体抑制。     

犬细小病毒胶体金检测试纸条的制备与初步评价

为快速检测及诊断犬细小病毒及其引起的传染性疾病,应用胶体金免疫层析技术．采用柠檬酸三钠还原法制备25nm的胶体金颗粒,标记抗犬细小病毒单克隆抗体CPV—F1株后包被于玻璃纤维膜作为金标垫,在硝酸纤维素膜上分别包被羊抗小鼠IgG二抗和抗犬细小病毒单克隆抗体CPV—B6株作为质控线和检测线,将吸水垫、硝酸纤维素膜、金标垫和样品垫分别粘贴于背衬板上制成犬细小病毒抗原胶体金检测试纸条。特异性试验及与血凝试验的对比结果表明,该试纸条具有良好的特异性和敏感性,与进口产品的总符合率为98％。本研究为进一步研发犬细小病毒检测试纸奠定了基础。     

PCR试剂盒在早期诊断犬细小病毒感染中的应用

利用PCR技术研制犬细小病毒的PCR诊断试剂盒.使用该试剂盒能特异地扩增含犬细小病毒的样品.同血凝试验(HA)及ELISA方法比较,对109份样品进行检测,显示该PCR试剂盒具有特异、灵敏等优点.适合于对早期感染细小病毒的宠物犬进行诊断,能提高临床诊断符合率,及时确定医疗方案.     

猫泛白细胞减少症病毒的血凝及血凝抑制试验检测

猫泛白细胞减少症病毒是由细小病毒引起的传染病,能感染各种猫科动物。通过血凝及血凝抑制试验(HA/HI),检测猫细小病毒的抗原和抗体效价,从而确定一种简易、快速操作简单、敏感、准确的诊断方法,并为免疫程序提供可靠依据。     

猪细小病毒血凝抑制试验抗原、阳性血清与阴性血清的制备与鉴定

猪细小病毒（Porcine Parvovirus,PPV）血凝抑制试验抗原、阳性血清和阴性血清在猪细小病毒制品的效力检验中不可或缺,对猪细小病毒相关生物制品的质量控制至关重要。试验采用PPV 7909株病毒同步接种PK-15细胞制备血凝抑制试验抗原,用制备的抗原乳化后免疫豚鼠制备阳性血清,同时用未免疫的阴性豚鼠制备阴性血清。对抗原、阳性血清和阴性血清进行鉴定,结果表明,制备的PPV血凝抑制试验抗原HA效价达1:512;阳性血清HI效价达1:1024;阴性血清HI效价＜1:8,且特异性均良好。利用制备的血凝抑制试验抗原、阳性血清与不同PPV疫苗株的灭活抗原、阳性血清进行交叉反应试验,结果表明,制备的抗原与阳性血清具备良好的血清学交叉反应性,可用于PPV制品的统一评价。     

驴抗犬细小病毒IgG的研制

用关中驴制备抗犬细小病毒（CPV）免疫球蛋白，预防和治疗犬CPV感染。制备CPV抗原复合物，通过免疫驴，用纯化方法从驴血清中提取特异性抗体（IgG），经过物理化学检定、血清学、免疫金电镜技术、动物效力实验。免疫驴均可产生特异IgG，免疫金电镜可观察到抗原抗体免疫复合物；血凝抑制试验（HI）≥1：512（IgG浓缩浓度为60mg/mL)；可保护犬免受CPV攻击；室温保存半年，4℃两年效价不变。已成功地应用CPV免疫驴制备出高效价特异性抗CPV－IgG。     

间接ELISA检测犬细小病毒病血清抗体方法的建立

用犬肾细胞增殖犬细小病毒,经纯化﹑标化作为包被抗原,建立检测犬细小病毒抗体的间接ELISA方法。结果表明:抗原最佳包被浓度5μg/mL;血清最佳稀释度1:40,反应时间45min;酶标抗体最佳稀释度1:2000,反应时间45min;S/P≥0.240判为阳性,≤0.190判为阴性,介于二者之间为可疑。该抗原不与犬瘟热﹑犬传染性肝炎﹑犬冠状病毒病﹑猫泛白细胞减少症病原阳性血清反应;批内重复试验变异系数小于10%,批间重复试验变异系数小于15%;对20份免疫犬血清进行检测,阳性检出率为85%,明显高于血凝抑制试验(65%),符合率为80%。试验结果表明建立的间接ELISA检测犬细小病毒抗体方法特异、灵敏、可重复性好。     

Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections

An enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specific mouse monoclonal antibodies, which recognise different epitopes of the haemagglutinin of CPV and which also neutralise the virus. A double antibody sandwich (DAS) ELISA for the detection of CPV in dog faeces was compared with the haemagglutination (HA) test. The DAS-ELISA proved to be more specific, sensitive and easier to perform than the HA assay. An indirect ELISA and a competitive ELISA for the detection of CPV-specific antibodies in dog sera were compared with the haemagglutination inhibition (HI) test. Both ELISA systems proved to be specific and easy-to-use methods for the detection of CPV-specific antibodies. The indirect ELISA, specially, proved to be more sensitive than the HI test. The higher sensitivity and specificity of the ELISA's as compared to HA and HI tests, and their ease of use, make them suitable for routine use in the serology and diagnosis of CPV infections.     

Evaluation of a new in‐clinic test system to detect feline immunodeficiency virus and feline leukemia virus infection

Background: Many in‐house tests for the diagnosis of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infection are licensed for use in veterinary practice. A new test with unknown performance has recently appeared on the market. Objectives: The aims of this study were to define the efficacy of a new in‐clinic test system, the Anigen Rapid FIV Ab/FeLV Ag Test, and to compare it with the current leading in‐clinic test, the SNAP Kombi Plus FeLV Antigen/FIB Antibody Test. Methods: Three‐hundred serum samples from randomly selected healthy and diseased cats presented to the Clinic of Small Animal Medicine at Ludwig Maximilian University were tested using both the Anigen Rapid Test and the SNAP Kombi Plus Test. Diagnostic sensitivity, specificity, and positive and negative predictive values were calculated for both tests using Western blot as the gold standard for verification of FIV infection and PCR as the gold standard for FeLV infection. Results: The presence of antibodies against FIV was confirmed by Western blot in 9/300 samples (prevalence 3%). FeLV DNA was detected by PCR in 15/300 samples (prevalence 5%). For FIV infection the Anigen Rapid Test had a sensitivity of 88.9%, specificity of 99.7%, positive predictive value of 88.9%, and negative predictive value of 99.7%. For FeLV infection, the Anigen Rapid Test had a sensitivity of 40.0%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 96.9%. Diagnostic accuracy was similar to that of the SNAP Kombi Plus Test. Conclusion: The new Anigen Rapid FIV Ab/FeLV Ag Test performed very well and can be recommended for use in veterinary practice.     

Hemagglutination by canine parvovirus: serologic studies and diagnostic applications

Conditions for canine parvoviral hemagglutination (HA) and hemagglutination-inhibition (HI) reactions were defined. The HA phenomena were used to differentiate canine parvovirus (CPV) from feline panleukopenia virus (FPV), mink enteritis virus (MEV), and minute virus of canines. Serologic comparisons of the CPV, FPV, and MEV by HA-HI and serum-neutralization tests indicated that CPV, FPV, and MEV were antigenically similar but were different from minute virus of canines. Diagnostic application of HA tests to fecal samples from acute cases of enteritis was discussed. Combinating HA tests with HI tests on fecal samples provided a rapid and specific diagnostic method for CPV infection. Secular seroprevalence studies indicated the emergence of CPV infeciton in the United States dog population-at-large in 1978.     

Evaluation of a gel diffusion precipitin test for porcine parvovirus

The use of a gel diffusion precipitin (GDP) test for the detection of porcine parvovirus (PPV) infection in pigs is described. The close correlation between gel diffusion precipitin and haemagglutination inhibiting (HI) antibody titres indicates that, with careful standardisation, a high level of sensitivity can be achieved with the GDP test and that it is a simple and relatively inexpensive alternative to the more commonly used HI test. Experimental infection of 2 groups of pigs showed that GDP and HI antibody responses were closely correlated and that GDP antibodies to PPV persisted for at least 41 weeks after infection. In a commercial herd study, serological evidence of declining passive immunity and subsequent acquisition of active immunity was demonstrated by measuring the GDP and HI antibody titres in sequential serum samples of pigs from a known PPV endemic farm. The GDP test described was shown to be less sensitive than haemagglutination (HA) in the detection of viral antigen but was, nevertheless, considered useful as a simple screening test for the amounts of antigen usually present in PPV infected mummified foetuses.     

同时检测犬瘟热病毒和犬细小病毒双重PCR方法的建立

为建立可以同时检测犬瘟热病毒(CDV)和犬细小病毒(CPV)的双重PCR方法,本研究根据GenBank登录的CDV N蛋白序列和CPV NS基因保守序列,设计合成2对特异性引物。通过优化反应条件,对CDV阳性病毒株反转录后的cDNA模板和CPV的DNA模板进行双重PCR扩增,同时得到2条与试验设计相符的669 bp(CDV)和392 bp(CPV)特异性条带,建立了同时检测CDV和CPV的双重PCR方法。实验结果表明:在同一PCR反应体系中可以同时检测这2种病毒,而对犬腺病毒Ⅰ型、犬腺病毒Ⅱ型、狂犬病毒检测均为阴性;CDV和CPV的最低检出限分别为101.8TCID50和101.4TCID50。采用该方法对在黑龙江省不同地区所采集的30份犬病料样品进行检测,CDV阳性率为30%;CPV阳性率为23.33%,表明建立的PCR方法可以用于临床诊断。     

犬细小病毒HZ0761株的分离与鉴定

采集疑似细小病毒(CPV)感染犬的粪便,采用同步培养法接种胎猫肾细胞(F81)进行病毒分离鉴定。通过PCR检测、HA试验、IFA鉴定、电镜观察和空斑纯化,获得1株犬细小病毒,并命名为HZ0761。感染的F81细胞48h后出现明显的细胞病变;在病料和感染的F81细胞中均扩增出CPV VP2基因的特异性片段(221 bp);病毒液可凝集猪红细胞,血凝价为1∶28,其血凝性能被特异性抗体抑制;IFA可见特异性亮绿色荧光;电镜观察感染的F81细胞核内可见20 nm左右的病毒颗粒;病毒液的TCID50为10-4.8/mL,VP2基因序列分析显示该毒株为CPV-2 a型。     

Establishment and Application of TaqMan MGB Fluorescent-Quantitative PCR Assay for Detection of Canine Parvovirus

In order to establish a TaqMan MGB fluorescent-quantitative PCR (FQ-PCR) assay for detecting canine parvovirus (CPV) specifically, sensitively and rapidly, a highly sensitive and specific TaqMan MGB FQ-PCR assay was developed using the specific primers and TaqMan MGB probe designed basing on the conservative sequences of VP2 gene of CPV in GenBank. The sensitivity, specificity and repetition assay of FQ-PCR assay were tested, and 46 clinic suspicious CPV infected samples were detected by the FQ-PCR assay in contrast to the routine PCR method. The results indicated that the FQ-PCR was successfully established. The developed FQ-PCR assay was able to detect as little as 1×10¹copies/μL of recombinant pGEX-T/CPV plasmid DNA, and the sensitivity of which was 100 times more than that of the routine PCR. The specificity assay exhibited that positive signals could be obtained from recombinant pGEM-T/CPV plasmid, but not from the genomic DNA or total cDNA of the other 5 kinds of pathogenic microorganism acting as the controls. The repetition tests were carried out by detection repeated 3 times for 3 different concentrations of recombinant pGEX-T/CPV plasmid, and the results indicated that the FQ-PCR was reproducible. Twenty-three positive results from 46 clinic suspicious CPV infected samples were obtained, which showed the better sensitivity than that of the routine PCR, with 19 positive samples from the same 46 suspected samples. The study suggested that the CPV FQ-PCR method was successfully established, and suitable for clinic rapid diagnosing of CPV and early detection of latent infection.     

应用多重RT-PCR方法检测158例猪粪样中的两种冠状病毒

根据GenBank上发表的猪流行性腹泻（PEDV）和猪传染性胃肠炎（TGEV）基因序列，针对其PEDVM（膜蛋白）基因保守区及TGEV的S基因（纤突蛋白基因）5’端保守区，各设计一对引物，可特异扩增出目的条带大小分别为467bp和1062bp。用上述两对引物对同一样品中的PEDV和TGEV可进行鉴别检测，对猪的其它病毒和细菌的PCR扩增结果均为阴性。敏感性测定结果表明：该双重PCR方法能检出PEDV1pg、TGEV0．1pg的模板。同时采用建立的多重RT-PCR及韩国引进的PEDV和TGEV病毒抗原快速诊断试剂盒检测结果显示：此多重RT-PCR方法特异性强，且较快速试剂盒更加敏感。应用多重RT-PCR对158份临床猪粪样的检测结果表明：我国很多猪场普遍存在TGEV和PEDV，尤其以PEDV污染更为严重，感染率达53．2％，但双重感染率较低，仅为4，4％。     

应用致敏SPA菌体提高CAV/CPV PCR检出率的研究

为消除粪便等病料中的PCR干扰物质 ,提高犬腺病毒与细小病毒 (CAV CPV)联合PCR检出率。根据免疫吸附的原理 ,应用CAV CPV高免血清致敏含A蛋白的金黄色葡萄球菌 ,制成致敏SPA菌体免疫吸附剂 ;以此免疫吸附提取粪便等病料中的CAV/CPV ,然后直接或经 3 5mol/LMgCl2 将所吸附病毒洗脱后再做PCR。结果 8份经电镜负染或HA/HI试验验证为阳性的粪便样品 ,如此处理后进行PCR检测 ,结果均为阳性 ;而直接取粪便离心后用上清进行PCR检测 ,结果均为阴性。表明该法可有效去除待检样品中的PCR干扰物质 ,提高PCR的检出率。     

Establishment and Application of Colloidal Gold Immune Chromatography Test Method for Detection of Canine Parvovirus Hemagglutination Inhibition Antibody Titer

The study was aimed to use colloidal gold immune chromatography technology to establish a rapid method for detection of canine serum canine parvovirus (CPV) hemagglutination inhibition (HI) titer and CPV vaccine immunization effect assessment.Double antibody sandwich method and monoclonal antibodies of anti-CPV hemagglutination antigen were used to prepare CPV antigen test strip.Canine serum with different proportion respectively was mixed with quantitative CPV antigen for full reaction,then dropped the mixture into the CPV colloidal gold test strip,so according to the highest serum dilution ratios when the test strip line T (line T) vanishes,it was to judge CPV antibodies in serum of the HI titer.This method had been used to detect 86 canine serum samples,at the same time,analyzing and comparing it with traditional hemagglutination inhibition test method.The results showed that the CPV antigen detection test strip was successfully prepared,and the reaction conditions and results of the test strip for detecting the titer of CPV-HI in canine serum were determined.The results indicated that when detecting CPV antigen after the dilution of different ratios of canine serum,the highest serum dilution ratios when the strip line T vanished and the HI titer had positive correlation.The highest dilution ratios of canine serum multiplied by 4 was the HI titer.The results of two methods had 90.7% consistency.This experiment established the colloidal gold immune chromatography test strip for the detection of CPV-HI titers method initially.This CPV-HI detection provided a simple and fast test method for the effect evaluation of CPV vaccine immune.