牛新孢子虫二温式PCR检测方法的建立与应用

根据已发表的犬新孢子虫种属特异性基因片段Nc-5基因序列设计合成一对特异性引物,采用均匀设计方法对引物浓度、Mg2+浓度、dNTP、Taq酶和退火温度进行了优化,建立了检测牛新孢子虫的二温式PCR方法.该方法对牛新孢子虫DNA的检测灵敏度为23.5 fg/反应.应用该方法对50份全血和8份流产胎儿样本进行了检测,有3份...     

牛新孢子虫内标双重荧光PCR检测方法的建立

为建立牛新孢子虫的快速准确检测方法,根据犬新孢子虫种属特异性基因Nc-5序列,设计高度保守的引物和荧光探针,通过引物设计和搭桥PCR法扩增,获得Nc-5荧光PCR内标模板。对内标模板的添加量和反应条件进行优化,建立了牛新孢子虫内标双重荧光PCR检测体系。该方法具有较好的特异性;可以检测到10个拷贝/PCR反应的核酸分子,与不加内标的荧光PCR检测灵敏度相当;通过对系列稀释的核酸样品的重复性检测,变异系数为0.50%～1.30%。通过对58份临床样品分别用该方法、不含内标的荧光PCR方法和普通PCR方法检测,结果显示,该方法与不含内标的荧光PCR方法的阳性检出率均为10.3%,比普通PCR方法阳性检出率（7.0%）高;表明该方法可用于临床样品中牛新孢子虫的快速检测,并能对实验室进行质量控制。     

弓形虫TaqMan-MGB荧光定量PCR检测方法的建立

为建立一种快速、敏感的弓形虫检测方法,本研究根据弓形虫GRA7基因保守序列设计特异性检测引物和TaqMan-MGB探针,建立了弓形虫荧光定量PCR检测方法。结果显示,该方法能特异地检测弓形虫DNA,而对新孢子虫、牛巴贝斯、马驽巴贝斯等虫DNA的检测均为阴性,具有良好的特异性;经3D数字PCR判定,其最低检测限为4.58拷贝/μL,灵敏度是常规PCR的1 000倍;重复性试验的组内、组间变异系数均小于5%;对62份疑似弓形虫感染流产的胎牛脑组织DNA进行检测,荧光定量PCR阳性检出率为24.19%(15/62),常规PCR为19.35%(12/62),阳性样品均包含于荧光定量PCR阳性样品中。本研究建立的荧光定量PCR检测方法可用于弓形虫病的早期诊断和日常监测,为监控弓形虫"带虫宿主"提供了良好的技术支持。     

实时荧光定量PCR检测柞蚕微孢子虫的方法

提高柞蚕微孢子虫检测技术的准确性和灵敏度,对控制柞蚕微孢子虫的胚种传染至关重要。以柞蚕微孢子虫小亚单位核糖体RNA(SSU rRNA)基因为靶基因,建立柞蚕微孢子虫的实时荧光定量PCR检测方法。结果显示:该方法能够特异性检测柞蚕微孢子虫基因组DNA,而对柞蚕基因组DNA无扩增产物。依据标准曲线确定荧光定量PCR的Ct值≤35作为检测的敏感度区间,对柞蚕微孢子虫基因组DNA的最低检出限为0.004 6 ng。应用该方法对100粒柞蚕蛹样本进行检测,共检出54个样本呈柞蚕微孢子虫感染阳性,而采用普通显微镜镜检与采用常规PCR方法检测呈阳性的样本数量分别为2个和34个。应用该方法对生产中的柞蚕种茧和雌蛾进行抽样检测,柞蚕微孢子虫感染阳性检出率显著高于用普通显微镜镜检的阳性检出率(P0.05)。建立的柞蚕微孢子虫实时荧光定量PCR检测方法提高了检测的灵敏度。     

布鲁氏菌和鹦鹉热衣原体双重PCR检测方法的建立与应用

为建立同时检测布鲁氏菌和鹦鹉热衣原体的双重PCR方法,本研究据GenBank上已发表的具有属间特异性的布鲁氏菌bp26基因和鹦鹉热衣原体23S rRNA基因,利用 Primer Premier 5.0软件各设计1对特异性引物,扩增的目的片段长度分别为219和356 bp。通过优化反应条件,建立了能同时检测布鲁氏菌和鹦鹉热衣原体的双重PCR方法。该方法具有较好的特异性和可重复性,对2种基因单重PCR检测敏感性均达到3.1×10²拷贝/反应,双重检测的灵敏度为3.1×10³拷贝/反应。利用该双重PCR方法对流产牛抗凝全血、血清、流产胎儿及奶液共172份临床疑似布鲁氏菌感染的样品进行检测,检测到布鲁氏菌阳性样品53份,鹦鹉热衣原体阳性样品2份,以上这2种病原的阳性检出率分别为30.8%和1.2%,且检测到2种病原混合感染的阳性样品2份,阳性检出率为1.2%。临床应用结果表明,该方法可用来对布鲁氏菌和鹦鹉热衣原体进行同步、快速、灵敏的检测。     

TaqMan荧光定量PCR在饲料沙门氏菌检测中的应用评估

为评估Taq Man荧光定量PCR应用于饲料沙门氏菌检测的可行性,本研究以沙门氏菌JEO402-1基因为靶基因,并采用已建立的沙门氏菌Taq Man荧光定量PCR检测方法,进行了特异性、灵敏度、检测限等相关方法学验证。结果显示:Taq Man荧光定量PCR法特异性良好,在饲料加标样本检测中的最低检测限为2×10~(-1)cfu/m L。重复性试验表明,在不同浓度样本中的变异系数均低于1.0%,具有较高的重复性和稳定性。利用该方法和传统培养法对72份饲料样本进行检测,结果显示两种方法检测结果符合率为65.3%,Taq Man荧光定量PCR法阳性检出率比传统培养法高34.7%;传统培养法检测阳性样本,PCR法检测结果均为阳性。以上结果表明,本研究建立的Taq Man荧光定量PCR法可以应用于饲料样本的沙门氏菌检测。     

流产衣原体TaqMan实时荧光定量PCR 检测方法的建立及初步应用

流产衣原体是导致绵羊地方性流产的主要病原体，给全球畜牧业经济发展构成了巨大威胁。为建立一种灵敏、特异且快速的检测流产衣原体的实时荧光定量PCR方法，依据衣原体蛋白酶样活性因子的基因组序列设计了针对检测流产衣原体的引物和TaqMan探针，对反应体系和反应条件进行了优化，对方法的灵敏度、特异性及重复性进行了评价，并初步应用于临床样本检测。结果显示，该方法的最低检测限为26 copies/μL，灵敏度是普通PCR的10倍；与其他可以引起类似症状的病原体无交叉反应；组内和组间变异系数均小于3%；对156份流产羊拭子的基因组进行检测，检出率为78.21%。研究表明，该方法可以很好的应用于流产衣原体的大规模临床样本检测，为流产衣原体病的高通量检测和流行病学调查提供技术手段。     

马疱疹病毒8型TaqMan实时荧光定量PCR检测方法的建立

为了建立一种能够快速检测马疱疹病毒8型(EHV-8)的方法，本试验以EHV-8的全基因组序列为模板，针对糖蛋白B(gB)基因的保守序列，进行对比分析，设计EHV-8的特异性引物和探针，优化反应条件，建立EHV-8 TaqMan实时荧光定量PCR检测方法，并对该方法的敏感性、特异性和重复性进行验证，使用该方法进行临床样本检测。结果显示，建立的EHV-8 TaqMan实时荧光定量PCR检测方法对EHV-8 DNA模板的最低检测限为1.1×10² copies/μL,敏感性高；EHV-8与EHV-1、EHV-4、马流产沙门氏菌和肠产毒性大肠杆菌均无交叉反应，特异性强；批内重复性试验和批间重复性试验均表明该方法重复性好。对132份临床样本的检测结果显示，阳性检出率为10.61%,基因测序正确。由此可见，本试验建立的EHV-8 TaqMan实时荧光定量PCR检测方法能够满足EHV-8的检测需求，且该方法敏感性高、特异性强、重复性好，为EHV-8的进一步研究提供了有效的辅助检测手段。     

检测鼻气管鸟疫杆菌SYBR GreenⅠ荧光定量PCR方法的建立

利用DNAStar对GenBank上发布的鼻气管鸟杆菌(ORT)16S rRNA基因进行序列分析,根据分析结果选择其保守区域设计1对特异性的引物。扩增16SRNA部分保守基因序列并将其克隆至pMD18-T载体,构建重组质粒,经鉴定、纯化后作为阳性标准品,用于SYBR GreenⅠ实时荧光定量PCR标准曲线的建立。反应条件反复优化后,成功建立了检测ORT的SYBR GreenⅠ实时荧光定量PCR方法。该方法对ORT的最低检测量约为100拷贝数/μL,比普通PCR高100倍;与引起禽类呼吸系统疾病的其他病原体之间无交叉反应;批内和批间试验的变异系数均小于0.63%,充分显示该方法具有良好的重复性和稳定性。对46份临床样本进行ORT检测,该方法检测的阳性样本为38份,检出率为82.26%。在38份阳性样品中,普通PCR只检出30份阳性样品,常规细菌培养只检出23份阳性样品。结果表明:本试验建立的SYBR GreenⅠ实时荧光定量PCR方法比其他方法更敏感,为临床上快速准确的检测、诊断禽类ORT的感染提供了敏感可靠的检测方法。     

犬细小病毒Taq Man荧光定量PCR检测方法的建立与初步应用

根据GenBank中CPV VP2蛋白基因序列,选择CPV VP2基因保守序列,分别设计1对特异性引物和TaqMan探针,建立了CPV Taq Man荧光定量PCR检测方法。对CPV Taq Man荧光定量PCR的反应体系和反应条件进行优化,并对其特异性和敏感性进行了测定,建立标准曲线。结果,CPV Taq Man荧光定量PCR对CPV检测阳性,并能对10拷贝/μL的CPV模板检测阳性,标准曲线的相关系数为0.9992,但对非CPV模板检测阴性。结果表明,所建立的CPV Taq Man荧光定量PCR具有很好的特异性和敏感性,标准曲线具有很强的实用性。同时对CPV Taq Man荧光定量PCR与CPV PCR进行了比较试验,结果前者比后者的敏感性高100倍。采用CPVHA、CPV PCR与CPV Taq Man荧光定量PCR对55份临床样品进行检测,CPV HA对30份样品检测阳性,CPVPCR对40份样品检测阳性,CPV Taq Man荧光定量PCR对47份样品检测阳性。这表明,CPV Taq Man荧光定量PCR对样品的检出率最高,具有特异、敏感、快速的特点,适用临床样品的检测。     

The importance of bovine neosporosis for abortion in Switzerland

The protozoan parasite Neospora caninum is one of the most important infectious abortion causes in cattle worldwide. In a Swiss case-control-study we investigated 113 abortion problem farms and 113 control farms. It was possible to detect N. caninum in 21% of 242 investigated aborted fetuses by PCR upon brain samples. Eighty-four % of mothers aborting N. caninum positive fetuses were serologically positive for this parasite. The seroprevalence for N. caninum of all aborting mothers was 44%. Within a period of 3 to 12 months 4,505 cattle from the participating farms were investigated serologically at two different time points. Strong fluctuations of the specific antibody concentration could be observed, resulting in a conversion to seronegativity in the second blood sample in 39% of formerly seropositive animals. Eighteen months after the end of the study, 42 case- and 42 controlfarms were questioned about their present abortion-status. Analysis of the questionnaire revealed that 80% of the former abortion problem farms observed an improvement of the situation. However, in 43% of those farms no preventive measures had been taken and no significant differences in hygiene, forage and animal turnover could be observed between case- and control-farms. Additionally, no significant differences were detected concerning risk factors such as the presence of farm dogs and the disposal procedure for placental and fetal material.     

Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1

Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.     

Potential involvement of Neospora caninum in naturally occurring ovine abortions in New Zealand

Neospora caninum is an obligate intracellular parasite and is recognised as the leading cause of bovine abortion worldwide. Natural infection with N. caninum has been described in sheep but it has generally not been regarded as a significant cause of abortion. Recently, there have been several New Zealand cases of foetal abortions where N. caninum was detected which strongly suggested the involvement of Neospora in these abortions. However, there is minimal information about the prevalence of N. caninum infection naturally occurring in New Zealand sheep flocks and particularly its impact on reproduction success. Thus, this present study provides preliminary data on the role that Neospora is playing in ovine reproductive failure by establishing the prevalence of N. caninum antibodies and DNA in ewe blood and foetal material present in 21 New Zealand sheep farms with ongoing unexplained abortion problems and 10 farms with consistently high fertility levels. The results of this study demonstrated an overall seroprevalence of 1.4% which varied between Aborting/non-pregnant (1.8%), age-matched pregnant controls (0.6%) and high fertility (2.1%) ewes. However, despite the variation observed, there was no statistical difference between the three groups. In addition, Neospora DNA was detected by PCR in 13% of submitted foetal brains and in ewe blood from aborting/non-pregnant (6.9%), age-matched pregnant controls (3.6%) and high fertility pregnant (2.1%) ewes. When the PCR results were considered with the IFAT and IDEXX ELISA results, there was no correlation between serology positive and PCR positive blood samples. Taken together, these results reveal that reliance on ELISA-based serology or PCR alone may underestimate the involvement of Neospora. Furthermore, determining the involvement of Neospora appears to require a multi-facetted approach where diagnostic methods and serological cut-off values may need to be adjusted as further information about the effect of natural infections with N. caninum in the ovine host is elucidated.     

新孢子虫dNcSRS2重组蛋白间接ELISA的建立及其应用

利用新孢子虫体外重组表面蛋白dNcSRS2蛋白作为包被抗原，对各项条件进行优化，确定判定标准，建立了检测新孢子虫血清抗体的间接ELISA方法。经对多例血清检测表明，所建立的诊断试剂盒重复性好、特异性强、灵敏度高，与进口的IFAT及两种商品化ELISA试剂盒的检测结果相比较，符合率均达到92％以上。应用建立的ELISA方法对236份奶牛血清的新孢子虫抗体进行检测，阳性率为22％。这是国内首次利用重组蛋白建立的诊断试剂盒，该方法的建立将为牛新孢子虫病的诊断与流行病学调查提供有效的技术手段。     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

Studies on the role of the red fox (Vulpes vulpes) as a potential definitive host of Neospora caninum

Neospora (N.) caninum is a protozoan parasite which is regarded as a major cause of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum which may shed environmentally resistant stages, oocysts, in their feces. Epidemiological studies in Germany showed that the presence of dogs increased the risk of a bovine herd to be N. caninum-positive in a bulk-milk ELISA test. However, there were also N. caninum-positive herds where dogs were not kept together with cattle.This leads to the question whether canids other than dogs, e.g., foxes, might be involved in the horizontal transmission of N. caninum. Therefore, the aim of our examinations in wild animals was to find out whether there are indications for a sylvatic cycle with foxes as definitive hosts and deer, roe deer and wild mice samples contained structures which resembled those of coccidian oocysts. In 13 of these 65 samples coccidian DNA was detected using a 18S rRNA gene based polymerase chain reaction (PCR).The examination of the 65 samples in a N. caninum-specific PCR revealed no positive result. Hammondia (H.) heydorni-DNA was detected in two samples. In addition, brain samples from 528 foxes, 224 wild mice, 16 deer and roe deer as well as from 1 wild boar were examined for the presence of N. caninum DNA by real time PCR. All samples tested negative by PCR. In conclusion, our study yielded no evidence indicating that the examined animals were part of a sylvatic cycle for N. caninum.     

Investigation for presence of Neospora caninum, Toxoplasma gondii and Brucella-species infection in killer whales (Orcinus orca) mass-stranded on the coast of Shiretoko, Hokkaido, Japan

Twelve killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait in Rausu, Shiretoko, Hokkaido, Japan on 8 February 2005. Tissue samples collected from 8 whales were tested for Neospora caninum, Toxoplasma gondii, and Brucella species DNA by polymerase chain reaction (PCR) assay. Gamma-globulin isolated from blood samples by ammonium sulfate precipitation was tested for antibodies to these pathogens by means of agglutination tests and immunoblotting. None of the 8 tissue samples had antibodies to the pathogens, when subjected to agglutination tests. In immunoblotting, one sample (sample No.5) showed antibody binding to N. caninum antigens. In the PCR assay, none of the samples was positive. Further study is necessary to examine the prevalence of the pathogens in marine mammals inhabiting this area.