A new molecular screening tool for the detection of chromosomal abnormalities in donkeys

Chromosomal abnormalities are a major cause of infertility and reproductive problems in equids. Nowadays, their detection is rising due to the use of new diagnostic tools based on molecular markers instead of karyotyping. Reports of this kind of genetic aberrations in domestic donkeys (Equus asinus) are extremely scarce, despite their importance in human activities. In the present study, we analysed the implementation of a short‐tandem‐repeat (STR)‐based molecular method initially developed for horses, as a diagnostic tool to detect chromosomal abnormalities in donkeys. The frequency of five X‐linked (LEX003, LEX026, TKY38, TKY270 and UCEDQ502) and one Y‐linked (ECAYM2) molecular markers and one Y‐linked gene (sex‐determining region Y, SRY) was characterized in 121 donkeys from two diverse breeds, the Spanish Andalusian and the African Moroccan breeds. The molecular panel showed 100% sensitivity and 99.67% specificity in detecting 10 different chromosomal abnormalities in the species. In conclusion, this methodology is a valid, rapid and low‐cost tool for the detection and characterization of chromosomal abnormalities in domestic donkeys.     

Evaluation of the suitability of monoclonal antibodies for flow cytometric intracellular cytokine detection in porcine peripheral blood lymphocytes

Panels of monoclonal antibodies (mAbs) specific for porcine interleukin (IL)-2, IL-4, IL-6, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were evaluated for their applicability in flow cytometric intracellular cytokine detection. Peripheral blood mononuclear cells were short-time stimulated in the presence of brefeldin-A, ionomycin and phorbol-12-myristate-13-acetate, fixed and incubated with the respective mAbs as well as phycoerythrin-conjugated second-step antibodies. Suitability of mAbs was judged by use of statistical data and by visual control of scattergrams, considering the capability of mAbs to discriminate between cytokine-positive and cytokine-negative cell populations. The number of suitable clones differed to a high degree between the investigated cytokines, but at least one mAb fitting for flow cytometric intracellular cytokine detection could be identified within each panel. Monoclonal Abs producing scattergrams with a clear demarcation between positive and negative cell populations were found within the anti-IL-2, IL-6 and IFN-gamma panels, whereas less well defined positive and negative cell populations could be generated by use of mAbs within the anti-IL-4 and TNF-alpha panels. Only one moderately fitting mAb was identified within the anti-IL-12 panel. After having evaluated the best fitting mAbs, these were used to obtain reference levels for the physiological range of porcine lymphocytic cytokine production in a second set of experiments. For that reason, 13 clinically healthy pigs aged between 6 weeks and 6 months were investigated. Data presented are given as mean +/- SD of the percentage of positive-staining lymphocytes: IL-2, 45.5 +/- 27.6; IL-4, 34.1 +/- 21.3; IL-6, 45.4 +/- 23.8; IL-12, 13.9 +/- 8.6; TNF-alpha, 43.4 +/- 11.3; IFN-gamma, 65.5 +/- 14.8.     

猪巨细胞病毒PCR诊断技术的建立

猪巨细胞病毒(porcine cytomegalovirus,PC-MV)属于疱疹病毒科、β疱疹病毒亚科、巨细胞病毒属的成员;近来有人认为将本病毒划在玫瑰疹病毒属中更为恰当.     

猪伪狂犬病病毒野毒和猪圆环病毒3型双重PCR检测方法的建立

为了能够同时检测和鉴别猪伪狂犬病病毒(pseudorabies virus,PRV)和猪圆环病毒3型(porcine circovirus type 3,PCV3)。本研究参考GenBank中公布的PRV gE基因和PCV3基因组序列,且基于PRV gE和PCV3 Rep基因的保守区域,利用Primer 5.0引物设计软件设计了2对特异性引物,随后对其扩增条件进行优化,建立了能够同时检测PRV和PCV3的双重PCR检测方法。该方法可以同时扩增PRV的429 bp和PCV3的344 bp特异性片段,而对猪圆环病毒2型、猪细小病毒、猪瘟病毒、猪繁殖障碍与呼吸综合征病毒、猪流行性腹泻病毒基因组扩增结果均为阴性。PRV和PCV3的最低检测值分别为502.0和91.2 copies/μL。结果表明该方法具有良好的特异性和灵敏性。本试验建立了能够同时快速检测PRV和PCV3,且具有高度特异性和灵敏性的双重PCR检测方法,为PRV和PCV3的检测提供技术支持。     

猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用

猪Delta冠状病毒(PDCoV)、猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)均是能引起仔猪腹泻的冠状病毒,通过临床症状和剖检很难鉴别诊断,建立同时检测且能够鉴别诊断这3种疾病的快速检测方法对于临床诊断具有重要的意义。参照GenBank中登录的PDCoV,NPEDV M和TGEV N基因的基因序列,利用Primer5.0设计合成3对能扩增特异性目的片段的引物,构建重组质粒,并对RT-PCR反应条件进行优化,建立了检测PDCoV,PEDV和TGEV的多重RT-PCR诊断方法,并对所建立的方法进行敏感性、特异性和重复性分析。利用该方法对河南176份临床样品进行检测,并与普通RT-PCR检测方法对该检测方法进行比较分析。结果表明,所建立的三重RT-PCR方法对PDCoV的最低检测量是3.14×103拷贝/μL;PEDV的最低检测量是3.88×104拷贝/μL和TGEV的最低检测量是3.68×104拷贝/μL。所建立的方法具有较好的特异性,对猪博卡病毒(PboV)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟(CSFV)和猪圆环病毒(PCV)等猪常见病毒的扩增结果均为阴性。应用所建立的三重RT-PCR方法对176份临床收集的仔猪腹泻样品进行PEDV,PDCoV和TGEV检测,并将检测结果和单重RT-PCR检测方法比较,结果显示,多重RT-PCR检测结果与常规单一RT-PCR的结果符合率均为100%。综上,本试验建立了能同时检测PDCoV,PEDV和TGEV的三重RT-PCR方法,为临床上这3种疫病的快速检测和流行病学调查奠定了基础。     

猪Hokovirus PCR检测方法的建立及其应用

猪Hokovirus(PHoV)是近年来在香港发现的一种猪细小病毒。为及时评估该病毒在我国的感染及流行情况,本研究根据GenBank中登录的PHoV核苷酸序列设计并合成一对特异性引物,建立了PHoV的PCR检测方法。研究结果表明,所建立的PCR检测方法特异性强,对其它的猪主要病毒无交叉反应;敏感性较高,最低可以检测2.4×104拷贝/μL;而且重复性较好。采用该方法对2009年～2010年我国华东地区猪场的318份临床样品进行检测,结果显示38份样品为PHoV阳性,表明我国猪群中存在PHoV感染。本研究建立的PCR方法可以作为在临床上检测PHoV的一种有效手段。     

Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen

Classical swine fever (CSF) is a highly contagious and severe viral disease of swine resulting in substantial production losses in different farming systems in many regions of the world. The accurate and rapid detection of CSF outbreaks is reliant on sensitive and specific laboratory testing and is a key component of disease control. Specific detection of CSF virus can be achieved by virus isolation in tissue culture, antigen capture or the detection of viral RNA using molecular techniques. In order to reduce the time taken to achieve a diagnostic result and simplify testing methods, an antigen capture ELISA using immunomagnetic beads (IMB) as the solid phase was developed and compared to a microplate-based antigen capture (AC)-ELISA. The IMB-ELISA has up to 64-fold greater analytical sensitivity than the AC-ELISA and initial estimates of diagnostic sensitivity and specificity are 100%. The IMB-ELISA has a highly robust, rapid and stable test format and is simpler to perform than the AC-ELISA. The IMB-ELISA has the added advantage that a result can be sensitively and specifically determined by eye, lending it to the possibility of adaptation to a near-to-field test with minimal equipment or expertise needed.     

Development of a bead-based multiplex PCR assay for the simultaneous detection of multiple Mycoplasma species

We describe the development and analytical validation of a 7-plex polymerase chain reaction assay coupled to a bead-based liquid suspension array for detection of multiple ruminant Mycoplasma spp. The assay employs a combination of newly designed and previously validated primer-probe sets that target genetic loci specific for Mycoplasma bovis, Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subspecies capripneumoniae (Mccp). Analytical sensitivity for the targeted Mycoplasma species ranged from 10 fg to 1 pg of purified gDNA extracted from broth cultures (approximately 8-800 MmmSC genome equivalents). In silico comparison of primers and probes, and analytical assessment with a range of near-neighbor Mycoplasma species and multiple bacterial respiratory pathogens demonstrated 100% analytical specificity of the assay. To assess assay performance and diagnostic specificity, 192 bovine respiratory samples were analyzed by incorporating a high throughput DNA extraction platform. The assay correctly classified all samples as negative for MmmSC or Mccp. All 33 field samples confirmed as positive for M. bovis by sequencing the uvrC gene were positive in the assay. The results from this study indicate that the bead-based liquid suspension array will provide a reliable, analytically sensitive and specific platform to simultaneously interrogate ruminant respiratory samples for multiple Mycoplasma species, including M. mycoides cluster organisms that are exotic to the United States. Sequential addition of primer-probe sets to the assay did not significantly impact analytical sensitivity of individual primer-probe combinations, suggesting that expanding the assay to include more Mycoplasma species will not compromise overall performance.     

猪胸膜肺炎放线杆菌PCR检测方法的建立及临床应用

为建立直接检测可疑病料中胸膜肺炎放线杆菌（APP）的PCR诊断方法，对用以扩增apxⅣA毒力基因3’端长约442bp的核苷酸片段的引物、模板、Taq DNA聚合酶、dNTPs的最适剂量及退火温度进行了优化筛选，并进行了特异性及重复性试验；以筛选出的优化条件，对临床可疑病料进行了PCR检测。结果，在25μL体系中，以5pmol／μL引物、0．25μL Taq DNA聚合酶、2mmol／L dNTPs、56℃退火温度对APP标准菌株apxⅣA毒力基因的扩增效果最好，重复性和稳定性高；而对类症菌株的扩增结果则为阴性，表明其特异性强。对分离到APP的病料PCR阳性率为100％（5／5）；未分离到APP的纤维渗出性病料中，新鲜与陈旧病料PCR阳性率分别为70．59％（12／17）、50．00％（4／8），而非纤维渗出性病料的PCR结果均为阴性（0／45、0／9）。结果表明，建立的PCR方法特异、快速、稳定、敏感，优于细菌学方法，它可作为APP可疑病料的理想诊断方法。     

3种猪冠状病毒抗体可视化检测芯片的研制与初步应用

猪的冠状病毒感染对于生猪养殖的危害性受到高度关注,其发病原因复杂多样,使得该类疫病的临床鉴别诊断成为一大难点。猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪血凝性脑脊髓炎病毒(PHEV)为3种常见的仔猪易感冠状病毒,对幼龄仔猪的致死率较高。本研究选取上述3种病毒核(N)蛋白进行原核表达与纯化,并将其固定至芯片载体上,经反应体系优化,制备了一种可同时检测上述3种病原特异性抗体的可视化蛋白芯片。将其初步应用于临床检测,结果表明,该标准化蛋白质芯片特异性、敏感性良好,最低检测限为稀释12 800倍,与ELISA的符合率在90%之上。本研究制备的可视化抗体芯片,能够迅速、高效、大量地对猪流行性腹泻、猪传染性胃肠炎、猪血凝性脑脊髓炎进行类症鉴别诊断,并有望应用于临床门诊及基层养殖场中PEDV、TGEV、PHEV 3种病原特异性抗体水平监测。     

猪捷申病毒TaqMan实时定量RT-PCR方法的建立

为建立猪捷申病毒(PTV)的早期检测及定量分析方法,本研究基于PTV 11个血清型基因组5′端非编码区保守序列,设计引物和TaqMan探针,建立了检测PTV的TaqMan实时定量RT-PCR方法.应用该方法对PTV、猪细小病毒、猪繁殖与呼吸综合症病毒、猪圆环病毒2型、猪伪狂犬病毒以及猪瘟病毒进行特异性试验,结果除PTV为阳性外其它均为阴性;针对PTV最低可检测到10个拷贝;批内、批间重复试验的变异系数均小于3％.应用建立的方法与病毒分离方法分别对91份临床样品进行检测,检出率分别为79.12％和57.14％,两者的符合率是78.02％.经临床应用表明,该实时定量RT-PCR方法可为PTV的早期诊断及定量分析提供技术手段.     

猪嵴病毒RT-PCR检测方法的建立及应用

根据GenBank中猪嵴病毒(porcine kobuvirus) 3D-RNA聚合酶基因序列设计并合成了1对特异性引物,建立了一种猪嵴病毒RT-PCR检测方法.反应产物测序结果与GenBank中猪嵴病毒SH-W-CHN/2010/China株比较,基因序列同源性为93％.利用该方法对猪蓝耳病毒、猪伪狂犬病病毒、猪瘟病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪轮状病毒、链球菌、猪巴氏杆菌和大肠杆菌等病原核酸进行扩增,均无条带出现,表明该方法具有较高的特异性;敏感性试验表明,该方法最低能检测到的病毒核酸含量为1 pg.利用所建立的RT-PCR方法检测采集自我国四川地区123份临床样品,结果阳性率为59.3％,表明猪嵴病毒在四川地区猪群中流行率较高.     

猪圆环病毒1型微滴数字PCR定量检测方法的建立

以猪圆环病毒1型ORF1基因为靶基因,建立了可对其准确定量的微滴数字PCR(droplet digital PCR,ddPCR)方法。对ddPCR反应中的探针浓度进行了优化。结果显示:ddPCR反应中的最佳探针浓度为250nmol/L;ddPCR方法线性相关系数(R2)为0.999,呈良好的线性关系;检测限为7.8copies/20μL;变异系数为2.7%;与其他病毒无交叉反应。通过对临床样品的检测,检测结果与实时荧光PCR结果一致。结果表明:新建立的ddPCR方法敏感性高、特异性强,可用于猪圆环病毒1型的定量检测。     

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.     

猪δ冠状病毒M基因RT-PCR检测方法的建立及应用

猪δ冠状病毒(PDCo V)是2014年在美国最新检测出的一种猪源冠状病毒。为评估PDCo V在我国的感染和流行情况,根据Gen Bank中登陆的PDCo V M基因核苷酸序列,设计合成1对特异性引物,建立了一种基于M基因的猪δ冠状病毒RT-PCR检测方法。研究结果表明,该方法特异性较好,仅对PDCo V有特异性的扩增,对其它主要猪病病毒核酸扩增均呈阴性;敏感性较高,最低检测限可达6.33×104拷贝/μL。运用该方法对我国华东地区猪场2014~2015年间的492份临床样品进行检测结果显示,PDCo V总阳性率为2.85%(14/492),其中腹泻样品中阳性率为18.60%(8/43),表明本研究建立的RT-PCR方法可以对临床样品进行有效检测。     

SYBR Green Ⅰ荧光定量PCR检测猪圆环病毒2型方法的建立

猪圆环病毒2型(PCV2)为圆环病毒科,圆环病毒属成员,是一种单股负链环状的DNA病毒,无囊膜,病毒粒子直径为17 nm.临床上对于该病毒相关疾病的诊断主要根据其临床症状,但由于猪圆环病毒同猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪细小病毒等引起的疾病临床症状相似,临床症状和病理变化只能作为初步诊断的依据,难以确诊.     

猪圆环病毒2型原位杂交检测方法的建立

参照GenBank发表的PCV2ORFl基因序列设计了1对引物,利用PCR地高辛探针合成的方法制备了长度为494bp的特异性探针,经检验具有良好的特异性和敏感性,可检测最低质粒DNA质量浓度为0．9728ug／L。用该探针建立了原位杂交组织切片检测方法,并用来检测PCV2感染猪的扁桃体和淋巴结组织,结果表明阳性信号主要存在于巨噬细胞胞浆中,信号强、背景良好,阴性对照无显色,说明该方法可作为PCV2实验室诊断和机理研究的一种有效检测方法。