PCR检测感染牛血液中边缘无浆体DNA

以边缘无浆体表面保护抗原的ｍｓｐｌβ基因设计和合成的１对２０ｍｅｒ寡核苷酸作为引物，用耐热的Ｔａｇ－ＤＮＡ聚合酶经５０个循环扩增边缘无浆体模板，扩增产物直接用凝胶电泳检测，并用标准分子量确定。结果表明，只有用边缘无浆体ＤＮＡ模板进行扩增时，扩增产物才能生成，而以血液原虫，如牛巴贝斯虫、双芽巴贝斯虫、锥虫和牛白细胞ＤＮＡ作模板扩增，无扩增产物生成。ＰＣＲ检测边缘无浆体的灵敏度可达约３００个无浆体的ＤＮＡ。边缘无浆体ＰＣＲ适用于牛边缘无浆体病的病因鉴定和检测     

牛呼吸道合胞体病毒一步法实时荧光定量PCR检测方法的建立及应用

为建立一种牛呼吸道合胞体病毒(Bovine respiratory syncytial virus, BRSV)快速简便的检测方法,本研究基于BRSV M基因保守区序列,设计特异性引物及探针,通过优化反应条件,建立用于BRSV检测的一步法实时荧光定量PCR,并验证了该方法的敏感性、特异性和重复性;同时利用建立的检测方法对采集的临床样本进行检测。结果表明：本研究所建立的BRSV荧光定量检测方法,其特异性好,仅对BRSV存在特异性扩增;敏感性高,最低可达10 copies/μL;稳定性好,组内变异系数和组间变异系数小。使用所建立的BRSV一步法实时荧光定量PCR对宁夏地区94份临床样品进行检测,阳性率为5.3%(5/94)。上述结果表明,本研究建立的检测方法可为BRSV的快速诊断提供有力的技术支持。     

嗜吞噬细胞无浆体SYBR GreenⅠ荧光定量PCR检测方法的建立及应用

为建立荧光定量PCR方法检测嗜吞噬细胞无浆体(Anaplasma phagocytophilum),针对GenBank A.phagocytophilum 16S rRNA基因保守序列(JN558815),设计1对引物1-25F/183-205R。以已知引物EE1/EE2和该引物进行巢式PCR扩增出预期大小片段(205bp),构建含有16S rRNA基因的重组质粒,以质粒为模板构建了标准曲线。以1-25F/183-205R为荧光定量PCR引物,建立A.phagocytophilum荧光定量PCR检测方法,并进行特异性、敏感性、重复性试验和临床样本检测。结果表明,该方法在6.4×10~3~6.4×10~8 copies/μL相关系数为0.99,显示出良好的线性关系;所建方法能特异地检出A.phagocytophilum,对绵羊无浆体、牛无浆体和环形泰勒虫基因组DNA和灭菌双蒸水均无交叉反应;可检测到6.4×10~2 copies/μL的标准质粒DNA,比常规PCR敏感性高100倍;批内及批间变异系数均低于2.0%,具有较高的重复性和稳定性;利用该方法对30份羊血液临床样品检出率比巢式PCR高16.67%。该研究为A.phagocytophilum临床检测和定量分析病原感染程度奠定理论基础。     

牛结节性皮肤病病毒TaqMan实时荧光定量PCR方法的建立与应用

牛结节性皮肤病(lumpy skin disease, LSD)是近年来我国新出现的一种牛传染性疾病。本研究根据LSD病毒(LSDV)的LSD022基因保守区域,设计出引物和探针,经过反应条件优化,建立了TaqMan实时荧光定量PCR检测方法,测试了其敏感性、特异性和重复性。结果发现,本方法敏感性达到15 copies/μL,不与其他牛病病毒和山羊痘弱毒疫苗AV41发生反应,变异系数均小于3%。用本方法和OIE推荐的普通PCR方法检测发病牛场94份临床样品,LSDV检出率分别为62%和37%,其中阳性样品2种方法检测符合率为96.7%,说明本方法敏感性、特异性和重复性较好,可用于LSDV检测。     

快速凝集试验与补体结合试验检测牛边缘无浆体感染的效果对比

从１３８份血清样品的比较试验结果显示，快速凝集成试验（ＲＣＡ）比补结合试验（ＣＦ）检测边缘无浆体感染的敏感性高（８８．９％：８１．５％），假阴性率低（１１．１％：１８．５％），两者都具有良好的特异性和预测性，检测阳性符合率高，快速凝集试验对一次感染牛的阳性时间更长久（３０３天：９２天）。     

牛支原体TaqMan探针实时荧光定量PCR检测方法的建立及应用

以牛支原体p80基因为靶基因,设计特异性引物与探针,优化反应体系,建立了牛支原体TaqMan探针实时荧光定量PCR(TaqMan real-time PCR)检测方法,并对该方法进行了特异性、敏感性、重复性评估以及临床样本检测。结果显示,特异性试验中,牛支原体2个菌株Cq值均小于18,其余13个非牛支原体菌株Cq值均大于36;敏感性试验中,最低可检3.89拷贝/μL靶DNA,是普通PCR的10~4倍(普通PCR 3.89×10~4拷贝/μL);重复性试验中,组内、组间重复的变异系数均小于1%;44份牛的临床样品利用TaqMan real time PCR检出6份阳性(阳性率为13.64%),分离培养鉴定检出7份阳性,普通PCR检测全部阴性。本研究建立了一种敏感、特异、稳定的牛支原体TaqMan real time PCR检测方法,可用于牛支原体的快速诊断和核酸定量检测。     

检测牛边缘无浆体抗体的竞争抑制ELISA方法的建立

为建立检测牛边缘无浆体(Anaplasma marginale)抗体的方法,本研究以牛A.marginale膜表面重组MSP5蛋白作为包被抗原,抗MSP5单克隆抗体(MAb)作为竞争抗体,建立一种用于检测牛A marginale抗体的重组MSP5蛋白竞争抑制ELISA(CI-ELISA)方法.经优化确定CI-ELISA的最佳反应条件为:抗原包被浓度为2μg/孔,封闭液为2%脱脂乳,MAb的稀释度为1:400,酶标二抗的稀释度为1:1000,阴性和阳性血清临界值分别为33%和40%;该方法具有良好的特异性和重复性;2 348份临床血清样品的检测结果表明,217份为阳性,阳性率为9.2%,与IDEXXA marginale抗体检测试剂盒的阳性符合率为95.3%,阴性符合率为100%.本实验建立的ELISA方法具有较高的特异性和重复性,可用于流行病学调查研究.     

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.     

牛结核分枝杆菌实时荧光定量PCR检测方法的建立及应用

为建立可应用于快速检测奶牛结核病、评估鲜乳污染状况、追溯传播途径的试验方法,本研究根据牛结核分枝杆菌基因组设计合成特异性引物,建立实时荧光定量PCR方法,并对反应条件进行优化,构建标准曲线,评价该方法的性能。结果显示,本研究所建立的实时荧光定量PCR方法能有效检测牛结核分枝杆菌目的基因,其最佳引物浓度为400 nmol/L,最佳退火温度为52 ℃。所构建的标准曲线相关性好,可用于样本的定量检测。该方法的性能评价显示,其最小检出模板浓度为80.24 ng/L,且该方法具有较好的特异性、可重复性,可对鲜乳样本进行检测。试验结果表明,本研究所建方法可用于牛结核分枝杆菌的定性和定量检测,这为奶牛结核病的诊断与净化及鲜乳食品安全评估提供重要技术。     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

边缘无浆体MSP5基因序列分析及其融合蛋白的纯化

参照已发表的主要表面蛋白5（MSP5）基因的核苷酸序列,设计了一对特异性引物,以边缘无浆体基因组DNA为模版。采用PCR技术扩增获得了MSP5基因;将其克隆到pGEM—TEasy载体,并进行测序分析,结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5基因的序列同源性达98．6％,编码氨基酸的同源性为99％,并且该序列包含有完整的开放阅读框,大小为633bp。将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体。将其转化到DH5a宿主茵中,用IPTG进行诱导表达,实现了融合表达。表达产物的分子质量为45ku。Western blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别。通过裂解、洗涤、变性、复性等方法对包涵体蛋白进行处理,获得的纯化产物浓度为1mg／mL。     

边缘无浆体MSP5蛋白基因的克隆及原核表达

参照已发表的边缘无浆体主要表面蛋白5（MSP5）基因的核苷酸序列,设计了1对特异性引物,以边缘无浆体基因组DNA为模板,采用PCR技术扩增获得了MSP5蛋白基因.将其克隆到pGEM-T Easy载体,并进行测序分析.结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5蛋白基因的序列同源性达98.6%,编码氨基酸的同源性为99.0%,并且该序列包含有完整的开放阅读框,大小为633 bp.将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体.将其转化到DH5a宿主菌中,用IPTG进行诱导表达,实现了融合表达,表达产物的分子质量为45 ku.Western-blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别.     

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.     

流产嗜衣原体TaqMan-MGB荧光定量PCR检测方法的建立

为建立一种快速、准确检测流产嗜衣原体(C.abortus)Taq Man-MGB荧光定量PCR方法,本研究根据C.abortus主要外膜蛋白基因的特异保守序列设计引物及探针,并优化反应条件,建立了检测C.abortus的荧光定量PCR方法。结果表明,以重组质粒为标准品建立的标准曲线在1.6×103拷贝/μL~1.6×107拷贝/μL内具有良好的线性关系,相关系数为0.9999。该方法仅对C.abortus的靶基因扩增呈阳性,而对鹦鹉热嗜衣原体、家畜嗜衣原体、鼠衣原体、沙眼衣原体、肺炎嗜衣原体、猪源衣原体核酸扩增结果均为阴性,特异性强;其最低检出限为1.6拷贝/μL;组内和组间重复性试验变异系数均小于3%,具有良好的重复性。利用建立的方法和普通PCR方法同时对225份临床样品进行检测,结果显示荧光定量PCR检出率比普通PCR高4.5%,表现较高的灵敏度和准确性。本研究建立的方法对C.abortus的临床鉴别检测和疾病诊断具有重要意义。     

Real-time PCR方法和PCR方法检测虾白斑综合征病毒

由白斑综合征病毒引起的虾白斑综合征是国际兽医局（OIE）规定的必须上报的水生动物二类疫病。本研究首先通过设计新的PCR引物，提高了PCR检测白斑综合征病毒的阳性检出率。为进一步提高白斑综合征病毒检测的灵敏度，本研究采用TaqMan探针技术建立了快速检测白斑综合征病毒的real-time PCR方法，通过与常规PCR方法比较，证实其检测灵敏度显著提高。     

Effect of breed of cattle on innate resistance to infection with Anaplasma marginale transmitted by Boophilus microplus

OBJECTIVE: To assess the innate resistance of and transmission in naive Bos taurus cross Bos indicus and purebred Bos indicus cattle when placed in a paddock with cattle infected with Anaplasma marginale and carrying Boophilus microplus ticks. DESIGN: A group of 49 purebred B indicus, and 48 B indicus cross B taurus (50%, F1 generation) 24-month-old steers were kept in the same paddock with cattle artificially infected with a virulent isolate of A marginale and Boophilus microplus. The cattle were seronegative for A marginale at the start of the trial but had previously been exposed to Babesia bovis and B bigemina. PROCEDURE: Cattle were inspected twice weekly for 118 days. Whole blood, blood smears and serum samples were collected from the cattle on day 37 after exposure and then at regular intervals to day 83 after exposure to measure packed-cell volumes, parasitaemias and antibody titres to A marginale. Any animals that met preset criteria were treated for anaplasmosis. On day 83 all cattle were treated with an acaricide and cattle infected with A marginale were removed from the rest of the group. RESULTS: A marginale was detected in blood smears from 14 crossbred and 9 B indicus steers between days 56 and 72 after exposure. Five and two of the infected crossbred and B indicus steers required treatment, respectively. One of the Bos indicus cattle died as a result of the A marginale infection despite treatment. Antibodies to A marginale were detected in the 23 infected cattle. The mean packed-cell volume depression was 40 and 37% in the affected crossbred and Bos indicus groups, respectively. There was no significant difference detected in susceptibility between these two groups. CONCLUSIONS: Innate resistance of purebred B indicus and crossbred cattle was not significantly different. The results confirm that purebred B indicus and crossbred cattle are sufficiently susceptible to warrant the use of vaccination against Anaplasma infections.     

Anaplasma marginale inactivated vaccine: dose titration against a homologous challenge

The present study was performed to dose-titrate an Anaplasma marginale experimental immunogen derived from partially purified initial bodies from three geographically different Mexican strains. Three five-bovine groups were inoculated twice on days zero and 21 with A. marginale initial bodies equivalent to 1.5×10¹⁰ (group I), 3×10¹⁰ (group II) or 6×10¹⁰ (group III) infected erythrocytes mixed with STDCM® adjuvant. A similar group served as non-vaccinated controls. All four groups were challenged with 1×10⁸ infected erythrocytes from a donor cow with an increasing rickettsemia of strain MEX-15 on day 87 post-vaccination. The prepatent period was very similar for all four groups. All five non-vaccinated controls presented typical acute anaplasmosis syndrome reaching a mean of 30.9% rickettsemia and a loss of 73.4% in the packed cell volume (PCV). Two of five controls died of acute anaplasmosis. Within the vaccinated groups only one animal (group II) suffered acute disease and died. Although all the other vaccinated animals were free of clinical signs, they developed very low rickettsemias (3.2, 3.8 and 4.3%) and PCV losses of 49.9, 47.8, and 49.3% for groups I, II and III. The starting mean weight was very similar for all four groups. All animals lost weight following challenge but losses for groups I and II were lower and significantly different from group IV losses (P0.1). Although there were no significant differences among vaccinated groups, group III was more severely affected. Taken altogether, these results show a 93.3% protection against both illness and death for all groups; and 100% protection for groups I and III, and 80% for group II.     

某羊场和田羊与策勒黑羊无浆体季节性感染PCR检测

为了解和田羊和策勒黑羊无浆体季节动态感染情况,分别于2015年冬至、2016年春分、夏至和秋分,在策勒县某牧业合作社采集和田羊和策勒黑羊血液样本共120份,应用PCR对无浆体的MSP4基因和16SrRNA基因进行扩增。结果发现,检测绵羊的无浆体总阳性率为99.2%(119/120),以绵羊无浆体(Anaplasma ovis)和嗜吞噬无浆体(A.phagocytophilum)混合感染为主,占阳性样本比例为61.3%(73/119),A.ovis和A.phagocytophilum占阳性样本比例分别为31.1%(37/119)和7.6%(9/119)。在不同季节,和田羊和策勒黑羊无浆体均普遍感染,差异不显著(P>0.05)。结果显示,新疆策勒县某羊场绵羊的无浆体病全年均可流行,应引起人们重视。