羊泰勒虫荧光定量PCR检测方法的建立

为建立一种敏感、快速的羊泰勒虫检测方法,本研究根据GenBank中登录的羊泰勒虫18S rRNA保守区设计1对特异引物,经各反应条件的优化,建立了羊泰勒虫荧光定量PCR检测方法。结果显示该方法可以特异地检测羊泰勒虫,而对卵形巴贝斯虫、双芽巴贝斯虫、羊巴贝斯虫和瑟氏泰勒虫检测均为阴性;该方法的灵敏度可达2.08×10~1拷贝/μL,比普通PCR灵敏度高100倍。组内及组间重复试验变异系数均小于5%。对50份临床样品进行检测,荧光定量PCR和常规PCR的阳性检出率分别为48%和38%。本实验建立的荧光定量PCR检测方法适用于羊泰勒虫定量分析和分子流行病学调查。     

梅迪-维斯纳病毒实时荧光定量PCR检测方法的建立

为建立检测梅迪-维斯纳病毒(MVV)的TaqMan实时荧光定量PCR方法,本研究根据MVV核苷酸保守序列设计引物和探针。以梯度稀释的含有MVV目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示:5.0×105～5.0×101拷贝范围内定量PCR均有S型扩增曲线,检测灵敏度为50拷贝。对羊的其他病毒核酸均无扩增反应。本研究建立的实时定量PCR方法,灵敏度高,特异性好,在MVV的快速检测中具有良好的应用前景。     

鸡毒支原体TaqMan实时荧光定量PCR检测方法的建立

为快速检测鸡毒支原体,根据鸡毒支原体的保守基因设计特异性引物和TaqMan探针,建立鸡毒支原体TaqMan实时荧光定量PCR方法并分析鸡毒支原体培养过程中颜色单位改变法(color change unit, CCU)和荧光定量PCR检测的相关性。结果显示,标准曲线y=-3.646x+44.208,相关系数R²=0.998,最低检测限度为1 copy/μL;与其他菌株等均无交叉反应;组内和组间变异系数均小于3%,表明该方法具有良好的稳定性和可重复性。用建立的实时荧光定量PCR检测方法对26份临床样品进行检测,该方法较普通PCR方法检出率更高。建立的荧光定量PCR与CCU显示鸡毒支原体在对数生长期时,两者具有一定的对应关系。表明建立了鸡毒支原体TaqMan探针实时荧光定量PCR检测方法,可实现对鸡毒支原体的快速检测,为鸡毒支原体感染的防控提供技术基础。     

牛巴贝斯虫实时荧光PCR检测方法的建立

为建立牛巴贝斯虫(B.bovis)的TaqMan实时荧光PCR检测方法,本研究根据GenBank中B.bovis的18S rRNA基因保守序列,设计引物和TaqMan探针,通过优化反应体系,建立检测B.bovis的实时荧光PCR方法.试验结果表明:实时荧光PCR对靶基因的最低检测值为1.31×101 copies/μL,比常规PCR的敏感性高1 000倍;而且与牛的其他血液原虫无交叉反应;组内及组间重复性试验的变异系数均小于3％,具有良好的重复性;在23份被检样品中,实时荧光PCR和常规PCR的检出率分别为52.17％和30.43％.该检测方法的建立为B.bovis的检测提供了一种快速、敏感、特异的技术手段.     

梅迪-维斯纳病毒实时荧光RPA检测方法的建立

为实现对梅迪-维斯纳病毒(MVV)的快速检测，根据MVV gag基因保守序列设计特异性引物和探针，通过优化反应条件和反应体系，建立了MVV的实时荧光RPA检测方法，并通过灵敏度、特异性、重复性试验及样品复核检测进行评价。结果显示：该方法最佳反应温度为39℃，用时短，20 min即可完成检测；灵敏度高，检测下限可达10^-1 pg/μL，比常规PCR检测结果灵敏约10⁴倍；特异性强，与小反刍兽疫病毒、羊败血性链球菌、牛肠道病毒、传染性鼻气管炎病毒、牛副流感病毒3型、肺炎支原体等继发呼吸系统症状病原无交叉反应；重复性好，对于相同质量浓度的模板DNA检测变异系数均小于10%；可有效检测出MVV，与PCR结果一致。综上所述，本研究建立的MVV荧光RPA检测方法适用于MVV的临床快速检测，可为该病的诊断及防控提供依据。     

羊口疮病毒TaqMan实时荧光定量PCR检测方法的建立及应用

根据GenBank中的羊口疮病毒基因组(AY386264)ORFVgORF121及ORFVgORF122基因的DNA序列,应用Beacon Designer 7.7软件设计一对引物和一条TaqMan探针,以含有该引物扩增序列的重组质粒作为阳性标准品,建立了羊口疮病毒TaqMan实时荧光定量PCR检测方法。该方法组内及组间重复试验变异系数均低于2%,上机检测时间不超过40min,检测灵敏度达1×101copies/μL,山羊痘及禽痘病毒特异性检测均为阴性,标准曲线的相关系数(R2)为0.998 088,线性关系良好。应用该方法对云南保山和普洱送检的羊口疮临床组织病料进行绝对定量检测,送检样品抽提DNA中病毒拷贝数分别为1.35×108copies/μL和9.62×107copies/μL。结果表明,研究建立的羊口疮病毒TaqMan实时荧光定量PCR检测方法具有特异、灵敏、稳定、快速和安全的特点,适于羊口疮临床组织样品的早期检测及常规病原监测。     

猪伪狂犬病病毒TaqMan荧光定量PCR方法的建立

针对猪伪狂犬病病毒(pseudorabies virus,PRV)gE基因保守区的核苷酸序列设计1对特异性引物和TaqMan探针,建立并优化了一种可快速、定量检测PRV野毒的TaqMan实时荧光定量PCR方法。应用该方法检测猪常见病毒性病原(猪瘟病毒、猪细小病毒、猪圆环病毒2型),PRV gE基因缺失株,以及健康猪的组织,结果均为阴性,证明该方法特异性良好。该检测方法能够检到的阳性质粒模板最低浓度为254 copies/μL,最低病毒浓度为4.22 TCID_(50)/100μL,比常规PCR敏感性高10倍;重复性试验结果表明该方法重复性良好;用TaqMan实时荧光定量PCR方法和常规PCR方法同时检测37份临床样品,其中前者检出阳性病料22份,阳性检出率为59.64%,后者检出阳性病料15份,阳性检出率为40.54%,2种方法的符合率为81.08%。综上所述,该方法的建立为PRV的实验室诊断及流行病学调查提供了快速、准确的检测手段。     

副猪嗜血杆菌及巴氏杆菌双重荧光定量PCR检测方法的建立

为建立副猪嗜血杆菌(Hps)、多杀性巴氏杆菌(Pm)的双重荧光定量PCR检测方法,本研究基于Hps的Omp P2基因,Pm的PlpE基因设计两对特异性引物及探针,通过对反应条件优化,建立了一种同时检测Hps及Pm的双重荧光定量PCR方法。该方法能够特异性地检测Hps和Pm,其对重组质粒标准品的最低检测浓度分别为5.60×10^2拷贝/μL、7.58×10^2拷贝/μL。双重与单一荧光定量PCR最低检测限相同,且均是常规PCR的100倍。重复性试验结果显示,该方法的组内和组间变异系数均小于2.5%。临床应用结果显示:该方法对阳性样品的检出率为53.57%,明显优于常规PCR和细菌分离鉴定。该方法能够用于两种疾病的同时检测和快速排查疾病。为两种疾病的防治提供有效检测工具。     

应用TaqMan荧光定量PCR检测H3N8亚型马流感病毒

为建立马流感病毒(ETV)的检测方法,本试验针对H3N8亚型ETV血凝素(HA)基因高度保守序列设计并合成了2对引物和1条TaqMan荧光探针,建立了TaqMan荧光定量PCR方法.经用TaqMan荧光定量PCR、RT-PCR和病毒分离方法分别检测新疆等省135份疑似EIV马鼻拭子样品,其结果表明:3种方法的马流感检出率分别为54.07%、37.78%、0.89%;TaqMan荧光定量PCR可检出马流感病毒基因组RNA的灵敏度可达10拷贝/反应.而且与其他马呼吸道病毒均无交叉反应,具有良好的特异性、敏感性和重复性.该方法为H3N8亚型马流感的早期诊断及分子流行病学调查等提供了一种新的快速、准确的定量检测技术.     

羊鞭虫病实时荧光PCR检测方法的建立

为建立一种快速检测羊鞭虫病实时荧光PCR方法,根据GenBank已经公布的羊鞭虫(Trichuris ovis)ITS基因序列(登录号:JF680987.1),设计特异性引物和TaqMan探针,以重组质粒作为绝对定量模板,建立检测羊鞭虫病的TaqMan实时荧光PCR方法。对反应体系的特异性、敏感性和稳定性进行评价,并用该方法对临床样品进行检测。结果显示,该检测方法线性关系良好,标准曲线的相关系数R^2=0.994,扩增效率E=1.05。该方法特异性强,与其他8种常见家畜寄生性线虫病不发生交叉反应;灵敏度高,最低检出下限为31.7拷贝/μL;重复性好,组内和组间变异系数分别为0.43%~1.04%和1.20%~1.91%,均小于2.00%。用建立的实时荧光PCR方法和显微镜检查方法分别对20份临床样品进行检测,实时荧光PCR和显微镜检查方法检出的阳性样品分别为14和9份。本研究建立的TaqMan实时荧光PCR方法能在粪便中快速、准确、灵敏检测羊鞭虫卵,为羊鞭虫病的检测和防控提供新的方法。     

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.     

Application of PCR technique in diagnosis of small ruminant lentivirus infection in sheep and goats

Detection of small ruminant lentiviruses (SRLVs) in sheep and goats usually relies on serological testing. In this study, we evaluated semi-nested PCR and nested PCR techniques applied as a diagnostic tool for detection of maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) in naturally infected sheep and goats, respectively. The examination of 193 ovine and 85 caprine serum samples by the ELISA revealed the presence of specific antibodies in 133 (69%) and 18 (21.2%) animals, respectively. Presence of proviral DNA was manifested in 103 (53.4%) sheep and 12 (14.2%) goats. Despite the relatively lower sensitivity of PCR, the fact of detection of proviral DNA in 19 out of 60 ovine samples and 7 out of 67 caprine samples collected from animals previously negative by ELISA was noteworthy. In conclusion, the data demonstrated that combinations of both ELISA and PCR might afford optimal detection of SRLVs infection.     

The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech Maedi-Visna isolates based on gag gene sequences

A semi-nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi-Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6%) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

The detection of proviral DNA by semi‐nested polymerase chain reaction and phylogenetic analysis of czech Maedi‐Visna Isolates Based on gag Gene Sequences

A semi‐nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi–Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6 %) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.     

猪伪狂犬病病毒野毒LNA探针real-time PCR检测方法的建立

本研究根据猪伪狂犬病病毒gE基因保守区域设计特异性引物和LNA-TaqMan探针,建立了基于LNA-TaqMan探针的猪伪狂犬病病毒野毒株荧光定量PCR检测方法.结果显示:所建立的方法能够特异性的检测出猪伪狂犬病病毒野毒株;灵敏度更高,最低检测下限为10个拷贝/μL;批内变异系数和批间变异系数分别为0.43％～0.64...     

牛轮状病毒三种核酸检测方法的比较

利用针对牛轮状病毒（BRV）的普通PCR、实时荧光定量PCR（Real-time PCR）和环介导等温扩增技术（LAMP）三种核酸检测方法对不同质粒浓度的样品和417份临床疑似样品进行检测,比较三种核酸检测方法的检出结果。三种核酸检测方法中LAMP最敏感敏感,能检测到1拷贝/μL,Real-time PCR能检测到100拷贝/μL,普通PCR只能检测到1×104拷贝/μL。但结合临床样品检测表明：常规PCR方法敏感度低会造成一部分漏检,LAMP灵敏度高又会造成错检。综合比较三种方法后,推荐用LAMP结合real-time PCR,不仅节约成本,且结果更为准确可靠,可提高牛轮状病毒的检出率。     

布鲁菌Cycling探针荧光定量PCR检测方法的建立

根据布鲁菌BCSP31基因序列设计布鲁菌通用检测引物和探针,建立了布鲁菌Cycling探针荧光定量PCR检测方法。以构建的含BCSP31基因的质粒标准品10倍递进稀释为模板检测其敏感性,结果显示,本方法能检测约10个拷贝的阳性质粒,且标准曲线的线性关系良好。用本方法检测5株不同种的布鲁菌以及猪大肠杆菌K99、巴氏杆菌C48-1、猪链球菌ST171、绿脓杆菌等4株对照菌。结果显示,5株不同种的布鲁菌均出现典型的"S"型扩增曲线,4株对照菌40个循环内均无CT值出现。用本方法和B4/B5-PCR方法对来自布鲁菌病流行地区3个不同牛场的40份血样、奶样和血清样进行平行检测。结果显示,本方法和B4/B5-PCR方法的结果符合率为80.0%。B4/B5-PCR检测为阳性的27份样品经本方法检测均为阳性;B4/B5-PCR检测为阴性的13份样本,经本方法检测,其中8份呈阳性,5份为阴性。本方法的敏感性明显高于B4/B5-PCR方法。试验表明,所建立的Cycling探针荧光定量PCR方法具有敏感、特异、稳定等特点,可用于布鲁菌感染的快速检测。     

反刍动物埃立克体荧光定量PCR检测技术的建立和应用

为快速、准确地检测反刍动物埃立克体,本研究以反刍动物埃立克体pCS20为靶基因设计特异性引物和探针,建立了TaqMan和Eva Green荧光定量PCR方法,对其反应的特异性、敏感性和重复性进行了分析,并与OIE推荐的套式PCR方法一起对临床样品进行检测。结果显示,本方法特异性强,与牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体无交叉反应;TaqMan和Eva Green荧光定量PCR对pCS20质粒标准品的最低检测限分别为17.4拷贝·μL^-1和1.74拷贝·μL^-1,标准曲线相关系数大于0.99,组内和组间CV均小于1.5%。对420只钝眼蜱样本的检测显示,TaqMan和Eva Green荧光定量PCR的检出率分别为25.48%和29.29%,与套式PCR检测方法相比,敏感性更高。本研究为反刍动物埃立克体的检测和流行病学调查提供了一种快速、准确的检测方法。     

猪链球菌2型荧光PCR检测方法与常规检测方法的比较

为验证猪链球菌2型荧光PCR检测方法对临床样品检测的敏感性和适用性以及该方法所拥有的独特优点,分别用荧光PCR法、常规PCR法和细菌分离法对人工感染猪链球菌2型的小鼠肝脏和发病猪的心、肝、脾、肾等实质器官和血液、喉拭子进行抗原检测。结果显示,荧光PCR法检出率为70.8%,明显高于普通PCR法（检出率为20.8%）,也高于常规细菌分离法（检出率为45.8%）。由于临床样品常常会被其他细菌污染,细菌分离法很难准确分离到链球菌。但荧光PCR法不受其他细菌污染的影响,对实验室培养的猪链球菌2型菌液,该方法检测滴度可达10-6/0.1mL（42～52 CFU/0.1 mL）,而普通PCR方法检测滴度仅为10-4。