运用双重PCR和ELISA法对云南部分地区PRRSV和PRV的检测分析

为了解云南省猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)和猪伪狂犬病病毒(Pseudorabies virus,PRV)的流行情况,试验采集云南省不同地区具有呼吸道症状的猪血清样品174份,首先建立PRRSV-GP基因和PRV-gB基因的双重PCR检测方法,然后运用建立的双重PCR方法和ELISA抗体检测试剂盒对PRRSV-GP和PRV-gB的阳性率及抗体水平进行检测分析。结果表明:试验建立了最低检测限度分别为1.48 ng/μL和0.67 ng/μL的PRRSV-GP和PRV-gB的双重PCR检测方法;单一PCR和双重PCR检测的PRRSV-GP阳性率为45.4%(79/174),PRV-gB阳性率为75.86%(132/174),混合检出率为5.17%(9/179);ELISA检测试剂盒检测的gE阳性率为0(0/174),PRRSV和PRV-gB的阳性率结果与单一PCR、双重PCR检测结果一致。说明试验构建的双重PCR方法有良好的敏感性和特异性;云南省部分地区PRRSV抗体阳性率低且免疫效果较差,PRV抗体阳性率相对较高且免疫效果良好。     

鉴别猪伪狂犬病病毒的双重PCR方法的建立及初步应用

为了检测猪伪狂犬病病毒(pseudorabies virus,PRV)的感染情况,并进行强弱毒株的鉴别诊断,本研究建立了快速、简便、灵敏度高、特异性强的鉴别猪PRV的双重PCR方法。针对PRVgE和gB基因序列,分别设计了2对特异性引物,通过对退火温度(50~60℃,按照1℃递增)、引物浓度(0.2~1.4μL,依次增加0.2μL)的优化,结果表明,双重PCR反应的最佳退火温度为56℃、最适引物添加量为1μL。特异性试验结果表明,该方法可以扩增出PRVgE(316bp)和gB(432bp)的目的片段,对PCV2、PTV、CSFV、PPV、PRRSV、大肠杆菌的DNA或cDNA均无扩增。敏感性试验结果表明,PRVgE和gB的最低核酸检出量分别为4.4×10³和3.3×10³拷贝/μL,与单一PCR方法的敏感性相近。应用该方法对广东、广西地区临床送检的56份组织样品进行检测,检测结果显示,强毒感染阳性率为53.6%(30/56),阴性率为46.4%(26/56),未发现有弱毒感染。     

牛传染性鼻气管炎病毒和牛病毒性腹泻病毒双重PCR检测方法的建立与应用

为建立一种针对牛传染性鼻气管炎病毒(infectious bovine rhinortracheitis virus,IBRV)和牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的双重PCR检测方法,根据GenBank中IBRV gB(UL27)和BVDV 5’-UTR基因序列分别合成特异性引物,优化反应条件,建立了能够同时检测IBRV和BVDV的双重PCR方法,并对其特异性、敏感性、重复性进行了测试。结果显示：该方法对IBRV、BVDV和IBRV/BVDV可分别扩增出500、198、500/198 bp的特异性目标条带,对牛呼吸道合胞体病毒、牛冠状病毒、牛细小病毒、牛纽布病毒和牛支原体检测结果均为阴性;对混合液中IBRV、BVDV的最低检测限分别为10⁴和10³ copies/μL;3次重复性试验结果一致。采用建立的方法对79份临床可疑牛血清样品进行IBRV、BVDV和IBRV+BVDV检测,阳性率分别为12.66%、17.72%和8.86%,与IBRV和BVDV单重PCR检测结果符合率分别为90.00%...     

猪重要病毒性病原多重PCR检测方法建立及应用

本试验针对当前对养猪业危害严重的猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、伪狂犬病病毒(PRV)和猪细小病毒(PPV),分别建立了检测CSFV和PRRSV的双重RT-PCR方法和检测PCV2、PRV及PPV的多重PCR方法。所建立的方法对CSFV、PRRSV、PCV2、PRV和PPV核酸的检出量分别为7.5 pg/μL、14.2 pg/μL、6.9 pg/μL、9.2 pg/μL和8.6 pg/μL。应用建立的方法分别对陕西省的陕南、陕北、关中等地区部分猪场采集的118份血清进行病毒检测。5种病毒的检出率分别是,CSFV为4.23%(5/118),PRRSV为22.01%(26/118),PCV2为15.25%(18/118),PRV为22.88%(27/118),从血清样品中未检测到PPV。本研究建立的检测RNA病毒双重RT-PCR和DNA病毒的多重PCR方法,为猪主要病毒其快速检测提供了可选择方法。     

猪瘟和猪伪狂犬野毒与其疫苗株多重PCR鉴别方法的建立

为建立一步法鉴别检测猪瘟病毒(CSFV)和猪伪狂犬病病毒(PRV)野毒株与其疫苗株感染的多重PCR方法(m PCR),本实验通过比对分析Gen Bank中登录的CSFV和PRV的相关基因序列分别设计可以区分CSFV与PRV及其疫苗株的5对特异性引物,建立了其多重PCR鉴别检测方法。结果表明,建立的多重PCR方法对CSFV和PRV扩增为阳性,而对PCV2、PRRSV、JEV和BVDV扩增结果均为阴性;最低核酸检测量分别为1.7×10~3拷贝/μL(CSFV野毒株)、9.9×10~3拷贝/μL(CSFV-C疫苗株)、1.3×10~3拷贝/μL Guizhou-DY)、6.9×10~3拷贝/μL(Bartha-K61)和5.5×10~3拷贝/μL(SA215)。采用该方法对134份可疑临床样品进行检测,结果表明CSFV和PRV疫苗株与野毒株在能繁母猪、育肥猪、保育猪和哺乳仔猪中的5重感染率分别为2.6%(1/38)、7.7%(2/26)、8.3%(3/36)和8.8%(3/34)。本研究建立的多重PCR方法为从病原学方面快速鉴别CSF和PR疫苗毒与野毒提供了新的技术支撑,为规模化猪场实现猪瘟与猪伪狂犬的净化提供一种新方法。     

基于重组酶介导等温扩增技术的羊口疮病毒核酸检测方法的建立及初步应用

为建立羊口疮病毒(Orf virus,ORFV)核酸的快速检测方法，本研究根据ORFV B2L基因保守序列设计特异性引物，通过优化反应温度与时间初步建立了基于重组酶介导等温扩增技术(RAA)的ORFV检测方法。优化试验结果显示：该方法在37℃反应40 min检测效果最佳。采用该方法对ORFV、山羊痘病毒、O型和A型口蹄疫病毒、小反刍兽疫病毒、山羊副流感病毒3型等临床常见症状相似的病毒核酸检测，结果显示：该方法除对ORFV的检测结果为阳性外，对羊的其他病毒的检测结果均为阴性，特异性较强。将质粒标准品10倍倍比稀释(1×10⁹拷贝/μL～1×10⁰拷贝/μL)后为模板，利用本研究建立的RAA方法检测，结果显示：该方法对ORFV质粒标准品的最低检测限为1×10⁴拷贝/μL，敏感性较高。利用本研究建立的方法对95份临床样品(15份组织样品和80份血液样品)检测，结果显示：该RAA方法能够对临床样品快速检测，组织样品和血液样品的阳性率分别为33.33%(5/15)和6.25%(5/80)，该检测结果与普通PCR检测方法的符合率均为...     

牛流行热病毒TaqMan实时定量PCR检测方法的建立及应用

通过对牛流行热病毒(bovine ephemeral fever virus,BEFV)中G片段保守区的分析,运用SnapGene设计探针、引物,并优化反应条件。运用最适条件对该方法的敏感性、特异性及重复性进行试验,并检测临床采集的样品。结果显示,本方法特异性较好,其对牛蓝舌病病毒、牛赤羽病病毒进行检测的结果为阴性。该方法线性关系较好,在10~5～10~(10)拷贝/μL相关系数达到0.998。该方法较为灵敏,可检测到10拷贝/μL。批内和批间分别重复3次试验得到的变异系数平均值结果较低(3%)。使用该方法对广东省兽医临床重大疾病综合防控重点实验室收集的109份牛抗凝血样品进行检测,阳性率为7%。本试验成功建立牛流行热病毒TaqMan实时定量PCR检测方法,并将该方法初步运用于牛流行热病毒的临床检测,为BEFV在临床上的快速检测提供更好的方法。     

水貂犬瘟热病毒微滴数字PCR检测方法的建立与应用

为建立检测水貂犬瘟热病毒(mink canine distemper virus, CDV)的微滴数字PCR(droplet digital PCR,ddPCR)定量方法，以便提供水貂CDV在诊断检测方面的技术支持，本研究根据实时荧光定量PCR(real-time quantitative PCR,qPCR)检测方法原理，建立了水貂CDV数字PCR方法，并优化了反应条件，评估了特异性、敏感性以及重复性。结果表明：ddPCR方法检测CDV的最适引物浓度为900 nmol/L,探针浓度为250 nmol/L,退火温度为55℃,升降温度为2.5℃/s,最低检测下限为4.4拷贝/μL;除CDV特异性扩增，其他常见病毒特异性检测结果均为阴性；重复性试验的变异系数均小于5%。采用该微滴数字PCR和荧光定量RT-PCR方法对30份犬、貂、狐、貉等组织(其中CDV阳性样品7份)样品进行检测，检测结果与临床检测结果相符。本研究建立的微滴数字PCR方法对CDV定量检测特异性强、灵敏度高，重复性好，可以用于水貂CDV临床样品的核酸检测，对水貂CDV发病早期的诊断提供新的定量检测方法。     

一种快速检测猪伪狂犬病毒野毒株的普通PCR方法的优化研究

为了快速、便捷运用普通PCR对猪伪狂犬病毒(Pseudorabies virus,PRV)野毒株进行检测,试验选取PRV g B、g E基因保守区序列,设计2对特异性引物,通过正交试验设计探索最优水平组合的PCR体系,建立鉴别检测PRV野毒株的普通PCR方法,并验证其特异性、敏感性、重复性、稳定性以及对临床样品的检测效果。结果表明:PCR体系的最优水平组合为Taq Mix 8μL,g B、g E引物的终浓度分别为0.2μmol/L和0.2μmol/L。建立的普通PCR检测体系仅对PRV野毒株呈双阳性,对疫苗株g B基因呈单阳性,检测猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪流行性腹泻病毒、猪乙型脑炎病毒、大肠杆菌等均显示阴性;敏感度介于1×103~1×104TCID50/m L病毒效价之间;重复性与稳定性良好;检测20份广东地区猪场临床疑似样品,疫苗毒株阳性率为25%,野毒株阳性率为10%,g B、g E单项PCR检测的符合率为100%。说明试验优化建立的普通PCR方法能对PRV野毒株进行快速鉴别诊断。     

猪流行性腹泻病毒和猪博卡病毒3/4/5型双重PCR检测方法的建立

为建立快速检测猪流行性腹泻病毒(PEDV)和猪博卡病毒(PBoV)3/4/5型的双重PCR方法,本研究根据Gen Bank中登录的PEDV ORF1基因序列和PBoV不同基因型VP1序列,设计2对特异性引物,通过对PCR扩增条件的优化,建立了能够同时检测PEDV和PBoV3/4/5型双重PCR方法,特异性检测结果显示该方法对猪细小病毒、猪圆环病毒2型、猪伪狂犬病毒、大肠杆菌、沙门氏菌、猪链球菌核酸扩增均为阴性;敏感性检测结果显示,对PEDV和PBoV3/4/5型重组质粒标准品的最低检出量分别为837拷贝/μL和1 000拷贝/μL;临床样品的检测结果显示,所建立的双重PCR方法可同时有效地检测出PEDV和PBoV3/4/5型混合感染及单独感染。本研究建立的双重PCR方法具有良好的特异性、敏感性、重复性,为快速、高效检测PEDV和PBoV提供了技术帮助。     

羊痘病毒的研究进展

从羊痘的流行特点、羊痘病毒基因组结构特征、热点蛋白研究、羊痘的诊断和检测、疫苗研究五个方面进行了较为详细的综述,以期为羊痘病毒的研究及预防控制羊痘提供参考。     

日本乙型脑炎病毒ELISA抗体检测试剂盒的研制及初步应用

采用日本乙型脑炎病毒单克隆抗体预包被酶标板,将纯化的日本乙型脑炎病毒(JEV)作为检测用抗原,利用包被捕获法建立了用于检测猪乙型脑炎抗体的间接ELISA法。采用建立的ELISA法对50份已知阴性血清样本检测,临界OD450nm值为0.343,ELISA与IFA对200份血清进行平行检测,总符合率为92.9%,与商品化的同类国产试剂盒的符合率为95%。与其他常见的猪病毒阳性血清抗体无交叉反应,2~8℃保存12个月稳定。研制的ELISA抗体检测试剂盒为临床JEV血清抗体检测及其疫苗的免疫效果评价提供了技术手段。     

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep

Objective To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.
Design A clinical and pathological study.
Procedure Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies.
Results All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection.
Conclusions The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.     

葡萄藤秆资源的开发及饲养试验

开发废弃的葡萄藤秆资源,加工调制后,作为粗饲料的一部分,用于全混合日粮中,对周岁绵羊进行30 d的饲养试验。在葡萄藤秆添加比例为15%或20%时,取得了日增重300 g左右的好成绩,为葡萄藤秆饲料资源的推广应用打下了良好的基础。     

柑橘衰退病毒研究进展

柑橘衰退病毒引起的柑橘衰退病是一种广泛分布于世界各柑橘主产区的重要柑橘病毒病害,对柑橘产业的为害极大。随着我国柑橘产业的快速发展,近年来柑橘衰退病在我国多个柑橘产区爆发,造成了严重的经济损失。本文就柑橘衰退病毒的起源、类型、传播方式、蛋白功能、病毒与寄主的互作关系、茎陷点症状形成的机理和防治方法作一综述,以期为今后有效防控柑橘衰退病提供参考。     

一种新发现的动物病毒——施马伦贝格病毒

从病原学、流行病学、临床症状以及诊断防治等方面对新发现的施马伦贝格病进行了介绍,为进一步研究该病毒、控制其传播等提供参考。     

Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.     

Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.     

Aleutian mink disease virus in furbearing mammals in Nova Scotia,Canada

Background

Aleutian mink disease virus (AMDV) is widespread among ranched and free-ranging American mink in Canada, but there is no information on its prevalence in other wild animal species. This paper describes the prevalence of AMDV of 12 furbearing species in Nova Scotia (NS), Canada.

Methods

Samples were collected from carcasses of 462 wild animals of 12 furbearing species, trapped in 10 NS counties between November 2009 and February 2011. Viral DNA was tested by PCR using two primer pairs, and anti-viral antibodies were tested by counterimmunoelectrophoresis (CIEP) on spleen homogenates.

Results

Positive PCR or CIEP samples were detected in 56 of 60 (93.3%) American mink, 43 of 61 (70.5%) short-tailed weasels, 2 of 8 (25.0%) striped skunks, 2 of 11 (18.2%) North American river otters, 9 of 85 (10.6%) raccoons, and 2 of 20 (10.0%) bobcats. Samples from six fishers, 24 coyotes, 25 red foxes, 58 beavers, 45 red-squirrels and 59 muskrats were negative. Antibodies to AMDV were detected by CIEP in 16 of 56 (28.6%) mink and one of the 8 skunks (12.5%). Thirteen of the mink were positive for PCR and CIEP, but three mink and one skunk were CIEP positive and PCR negative. Positive CIEP or PCR animals were present in all nine counties from which mink or weasel samples were collected.

Conclusions

The presence of AMDV in so many species across the province has important epidemiological ramifications and could pose a serious health problem for the captive mink, as well as for susceptible wildlife. The mechanism of virus transmission between wildlife and captive mink and the effects of AMDV exposure on the viability of the susceptible species deserve further investigation.