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为了比较研究3种猪肺炎支原体(Mhp)的检测方法,利用已建立的显色原位杂交(CISH)和量子点荧光原位杂交(QD-FISH),并结合PCR方法,检测人工感染Mhp的试验猪、自然感染Mhp的试验猪和疑似感染Mhp的临床样品.结果显示,3种检测方法对人工感染组和自然感染组的检测率均为100％;对临床样品,PCR的检测率为90％,CISH的检测率为70％,QD-FISH的检测率为75％.结果表明,用于检测Mhp感染,PCR是最佳检测方法,但CISH和QD-FISH在Mhp检测和致病机理研究方面具有一定的应用价值. 相似文献
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利用生物素化的猪肺炎支原体核酸探针和量子点荧光显色系统,对人工感染组、自然感染组和健康对照组的试验猪肺样品,以及临床样品进行量子点荧光原位杂交检测。结果表明：按照探针质量浓度为1mg／L、80～90℃变性10min、37℃杂交10h和QDs—SA复合物浓度为10nmol／L的条件进行猪肺炎支原体的量子点荧光原位杂交检测结果比较理想;而人工感染组、自然感染组和健康对照组样品的QD-FISH检测结果与分离培养结果和PCR检测结果相比,符合率均达到100％刘占床样品的QD-FISH检测结果与PCR检测结果相比,符合率为83．3％。从而证明猪肺炎支原体量子点荧光原住杂交法是一种直观和敏感的检测方法,可以用于猪肺炎支原体的检测、定位和致病机理研究,也为其他病原的研究提供了参考方法。 相似文献
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应用原位杂交技术检测感染中国对虾和克氏原鳌虾体内的白斑综合征病毒 总被引：3，自引：0，他引：3
用PCR制备地高辛（DIG）标记探针，采用原位杂交技术检测感染中国对虾和克氏原鳌虾体内的白斑综合征病毒（WSSV）。患病严重的淑死中国对虾的角化上皮、胃上皮、触角腺、肝胰腺上皮、疏松结缔组织、肌肉、造血组织、鳃、卵巢之结缔组织细胞和滤泡细胞、精巢之结缔组织细胞原位杂交呈阳性；人工注射感染克氏原鳌虾的角化上皮、胃上皮、肝胰腺上皮、疏松结缔组织、肌肉、造血组织、鳃原位杂交检测呈阳性。PCR制备DIG标记探针与健康中国对虾和克氏原鳌虾无交叉反应，相应样品未加入探针杂交和未加入DIG抗体杂交均为阴性反应。 相似文献
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本试验旨在建立一种针对猪圆环病毒2型(PCV2)和猪肺炎支原体(Mhp)的双重PCR检测方法.对猪伪狂犬病毒(PRV)、猪细小病毒(PPV)、沙门菌、大肠杆菌、猪链球菌、副猪嗜血杆菌、巴氏杆菌、支气管败血波氏杆菌基因组模板进行PCR特异性检测,没有任何非特异性扩增.敏感性试验显示,建立的PCR检测方法能够检测到的PCV2和Mhp模板的最低浓度分别为130和180fg·mL-1.同时用单项PCR和双重PCR对湖北省的各大猪场的174份PRDC样品进行检测,PCV2和Mhp的符合率分别达到100％和98.28％.进一步应用双重PCR调查PCV2和Mhp在不同猪场的感染动态,结果显示有一定比例的仔猪在产房就已经感染PCV2和Mhp,大部分猪是在保育和育肥阶段被感染 ;但是不同猪场表现出不同的感染动态.本试验建立了一种针对PCV2和Mhp的双重PCR检测方法,具有良好的特异性、敏感性,为临床疾病检测和疾病流行态势调查提供了有力的支持. 相似文献
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《畜牧兽医学报》2020,(6)
旨在采用环介导等温扩增(LAMP)和横向流动试纸条(LFD)相结合的方法,建立一种快速、特异,过程可视化的猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)检测方法。针对猪肺炎支原体(Mhp)P36基因设计5套特异性引物和1条异硫氰酸荧光素(FITC)标记的探针,进行LAMP扩增反应。将生物素标记的LAMP产物与FITC标记的探针进行特异性杂交,并使用横向流动试纸条完成扩增产物的检测。经优化,LAMP最佳反应条件为65℃,反应15 min,从基因组DNA提取到LFD结果判断只需40 min左右,比常规PCR技术缩短近2 h。LAMP-LFD可特异性地检出猪肺炎支原体(Mhp),对猪鼻支原体(Mhr)及猪圆环病毒(PCV)等常见猪病病原的检测结果为阴性。灵敏性试验表明,LAMP-LFD对Mhp的检测灵敏度为1×10~0个拷贝DNA,是普通PCR的1 000倍。利用本方法可从采集的88份临床疑似病料样品中检测到64份阳性,国标PCR方法可检测到56份阳性,符合率可达90.9%。综上,本研究建立的LAMP-LFD方法可特异、准确地应用于Mhp的检测,而且灵敏度高、操作简单、仪器设备依赖性低、检测成本低、耗时短,适合基层实验室、应急检测或现场监测等使用,具有较高的推广价值,有望发展成为Mhp快速检测的有效手段。 相似文献
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猪支原体肺炎LAMP-LFD快速检测方法的建立及初步应用 总被引：1，自引：0，他引：1
旨在采用环介导等温扩增（LAMP）和横向流动试纸条（LFD）相结合的方法，建立一种快速、特异，过程可视化的猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）检测方法。针对猪肺炎支原体（Mhp）P36基因设计5套特异性引物和1条异硫氰酸荧光素（FITC）标记的探针，进行LAMP扩增反应。将生物素标记的LAMP产物与FITC标记的探针进行特异性杂交，并使用横向流动试纸条完成扩增产物的检测。经优化，LAMP最佳反应条件为65℃，反应15 min，从基因组DNA提取到LFD结果判断只需40 min左右，比常规PCR技术缩短近2 h。LAMP-LFD可特异性地检出猪肺炎支原体（Mhp），对猪鼻支原体（Mhr）及猪圆环病毒（PCV）等常见猪病病原的检测结果为阴性。灵敏性试验表明，LAMP-LFD对Mhp的检测灵敏度为1×100个拷贝DNA，是普通PCR的1 000倍。利用本方法可从采集的88份临床疑似病料样品中检测到64份阳性，国标PCR方法可检测到56份阳性，符合率可达90.9%。综上，本研究建立的LAMP-LFD方法可特异、准确地应用于Mhp的检测，而且灵敏度高、操作简单、仪器设备依赖性低、检测成本低、耗时短，适合基层实验室、应急检测或现场监测等使用，具有较高的推广价值，有望发展成为Mhp快速检测的有效手段。 相似文献
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猪支原体肺炎活疫苗(168株)是一种以肺内注射途径免疫的弱毒活疫苗。为了拓展猪支原体肺炎活疫苗的免疫途径,评估猪支原体肺炎活疫苗(168株)配合佐剂以肌肉注射方式免疫猪群后的攻毒保护效果,选取20头7日龄猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)阴性仔猪,将其随机平均分成4组,分别为健康对照组、感染对照组、肌肉注射免疫组和肺内注射免疫组。在免疫后采集血样并检测其中的Mhp IgG抗体,在首次免疫后42 d人工感染Mhp组织毒(JS株),攻毒28 d后评估肺脏的病变情况并测定支气管肺泡灌洗液(BALF)中的Mhp含量。结果显示：免疫后肌肉注射免疫组动物产生了明显的Mhp特异性血清IgG抗体,而肺内注射免疫组动物在攻毒前未见明显的血清抗体;肌肉注射免疫组和肺内注射免疫组的攻毒保护率分别平均为88.89%和75.93%,且组间无显著性差异;感染对照组的BALF中Mhp单位含量极显著高于肌肉注射免疫组和肺内注射免疫组(P<0.01),2个免疫组间无显著性差异。结果表明：猪支原体活疫苗配合佐剂后经肌肉注射免疫可产生较好的免疫攻毒保护效果。本研究为猪支原体肺炎活... 相似文献
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旨在建立猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)抗体胶体金免疫层析检测方法,从而快速地对Mhp的感染和免疫情况进行监测,预防和控制猪支原体肺炎传播。通过柠檬酸三钠还原法制备胶体金颗粒,确定Mhp特异性抗原P46蛋白的最适标记pH和标记浓度并进行标记,作为金标液。将P46蛋白包被检测线T线,抗P46蛋白单抗包被质控线C线,组装检测试纸条。经优化,建立可同时用于Mhp血清抗体和黏膜抗体检测的胶体金免疫层析检测方法。利用该检测方法对76份临床血清样品和40份临床鼻拭子样品进行检测,并与ELISA检测结果进行比较。用该试纸条检测血清样品时,灵敏性、特异性、重复性较好,与商品化Mhp ELISA抗体检测试剂盒符合率为96.1%;该方法检测呼吸道鼻拭子样品时,存在微弱的非特异性反应,检测结果与本实验室建立的sIgAELISA检测方法符合率为87.5%。本研究成功建立了Mhp抗体胶体金免疫层析检测方法,整个过程只需10 min。检测血清样品具有较好的特异性和灵敏性,检测鼻拭子样品时特异性还有待提高。 相似文献
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通过建立能对石蜡切片中鹅细小病毒(GPV)核酸进行定位的原位PCR方法,为GPV在鹅体内的定位、致病机理研究等提供有效的试验手段.根据GPV的VP3基因序列设计PCR引物和寡核苷酸探针,以GPV感染鹅肝脏和空肠组织石蜡标本制作切片,经蛋白酶K消化、原位PCR扩增和碱性磷酸酶标记的寡核苷酸探针原位杂交,建立了检测石蜡标本中GPV的间接原位PCR方法并应用于自然感染GPV高免血清紧急免疫后鹅肝脏和空肠组织临床病料检测.结果显示间接原位PCR对人工感染GPV死亡鹅肝脏和空肠的石蜡标本检测结果为阳性,而鹅病毒性肝炎、鹅多杀性巴氏杆菌病、鹅沙门菌病和鹅大肠杆菌病死亡鹅肝脏的石蜡标本检测结果为阴性;间接原位PCR对自然感染GPV高免血清紧急免疫后第6天的鹅肝脏和空肠组织检测20个样本中,空肠组织有8个呈阳性,肝脏组织有4个阳性结果均为阳性,阳性细胞有空肠上皮细胞、肝窦上皮细胞等. 相似文献
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Localization of swine influenza virus in naturally infected pigs 总被引：4，自引：0，他引：4
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根据猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)的16S rRNA基因设计3条引物, 建立Mhp和Mhr的双重PCR检测方法,并对该方法进行了特异性和敏感性试验,并使用建立的方法检测了临床样品和疫苗样品。结果显示该方法具有良好的特异性,最低可检测到0.66ng 的Mhp基因组DNA和0.58 ng Mhr基因组DNA,临床样品和疫苗样品检测结果与普通PCR检测结果一致。该双重PCR方法,可用于Mhp与Mhr的鉴别、诊断以及疫苗纯粹性检查,快速而准确。 相似文献
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Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection. 相似文献
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为探明姜曲海猪感染猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）后肺组织环状RNA （circular RNA,circRNA）差异性表达谱及其在抗Mhp感染中的作用,试验以姜曲海猪为研究对象,分为感染组和对照组,人工感染Mhp 28 d后,解剖采集肺组织,采用高通量测序和生物信息学软件分析circRNA的表达情况。测序数据比对参考猪基因组序列,共鉴定到23 632个circRNAs,差异表达circRNAs为213个,其中97个上调,116个下调。随机选择4个差异表达circRNAs进行实时荧光定量PCR验证,检测结果与测序结果基本一致。差异表达circRNA来源基因可注释到包括抗原加工递呈、溶酶体、白细胞跨内皮迁移等免疫应答信号通路。circRNA-miRNA-mRNA靶标关系分析显示,筛选到海绵结合miRNA数量最多的3个差异表达circRNAs,分别为：circRNA-17284（21个）、circRNA-04848（19个）和circRNA-17270（19个）;预测到6个与免疫调控相关的靶向miRNAs,分别为：ssc-miR-4331、ssc-miR-370、ssc-miR-328、ssc-miR-30c-3p、ssc-miR-122和ssc-miR-125b。本研究测定了感染Mhp的猪肺组织circRNA表达谱,筛选到与免疫调控相关的差异表达circRNA,这有助于阐明姜曲海猪对Mhp的易感机制,为抗病育种研究提供了参考依据。 相似文献
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Cyclooxygenase-2 (COX-2) was detected and localized in 15 pigs with naturally occurring pleuropneumonia using a 437-base pair digoxigenin-labeled cDNA probe in an in situ hybridization protocol. Histopathologic changes in the acute stage were characterized by coagulative necrosis of lung parenchyma, hemorrhage, vascular thrombosis, edema, fibrin deposition, and infiltration of lung parenchyma by neutrophils and alveolar macrophages in nine pigs. In chronic lesions, a thick layer of granulation tissue surrounded foci of pulmonary necrosis in six pigs. All 15 pigs infected with Actinobacillus pleuropneumoniae, confirmed by bacterial isolation, had distinct positive hybridization signals for COX-2 in bronchial, bronchiolar epithelial cells, alveolar macrophages, neutrophils, and type I pneumocytes. COX-2 expression was detected primarily in neutrophils from pigs with acute lesions and primarily in alveolar macrophages from pigs with chronic lesions. The results suggest that a prostanoid product of COX-2 is an important component of the inflammatory response to acute and chronic A. pleuropneumoniae infection. 相似文献
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K E Johansson J G Mattsson K Jacobsson C Fernandez K Bergstr?m G B?lske P Wallgren U B G?bel 《Research in veterinary science》1992,52(2):195-204
Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs. 相似文献
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Deblanc C Gorin S Quéguiner S Gautier-Bouchardon AV Ferré S Amenna N Cariolet R Simon G 《Veterinary microbiology》2012,157(1-2):96-105
Swine influenza virus (SIV) and Mycoplasma hyopneumoniae (Mhp) are widespread in farms and are major pathogens involved in the porcine respiratory disease complex (PRDC). The aim of this experiment was to compare the pathogenicity of European avian-like swine H1N1 and European human-like reassortant swine H1N2 viruses in na?ve pigs and in pigs previously infected with Mhp. Six groups of SPF pigs were inoculated intra-tracheally with either Mhp, or H1N1, or H1N2 or Mhp+H1N1 or Mhp+H1N2, both pathogens being inoculated at 21 days intervals in these two last groups. A mock-infected group was included. Although both SIV strains induced clinical signs when singly inoculated, results indicated that the H1N2 SIV was more pathogenic than the H1N1 virus, with an earlier shedding and a greater spread in lungs. Initial infection with Mhp before SIV inoculation increased flu clinical signs and pathogenesis (hyperthermia, loss of appetite, pneumonia lesions) due to the H1N1 virus but did not modify significantly outcomes of H1N2 infection. Thus, Mhp and SIV H1N1 appeared to act synergistically, whereas Mhp and SIV H1N2 would compete, as H1N2 infection led to the elimination of Mhp in lung diaphragmatic lobes. In conclusion, SIV would be a risk factor for the severity of respiratory disorders when associated with Mhp, depending on the viral subtype involved. This experimental model of coinfection with Mhp and avian-like swine H1N1 is a relevant tool for studying the pathogenesis of SIV-associated PRDC and testing intervention strategies for the control of the disease. 相似文献
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Mostegl MM Wetscher A Richter B Nedorost N Dinhopl N Weissenböck H 《Veterinary parasitology》2012,183(3-4):209-214
In this retrospective study 102 cats were analyzed for the presence of trichomonads in intestinal tissue sections using chromogenic in situ hybridization (CISH). Two intestinal trichomonad species are described in cats: Pentatrichomonas hominis and Tritrichomonas foetus. While P. hominis is considered a mere commensal, T. foetus has been found to be the causative agent of feline large-bowel diarrhea. For the detection of both agents within intestinal tissue CISH assays using three different probes were performed. In the first CISH run a probe specific for all relevant members of the order Trichomonadida (OT probe) was used. In a second CISH run all positive samples were further examined on three consecutive tissue sections using the OT probe, a probe specific for the family of Tritrichomonadidae (Tritri probe) and a newly designed probe specifically detecting P. hominis (Penta hom probe). In total, four of the 102 cats were found to be positive with the OT probe. Thereof, one cat gave a positive reaction with the P. hominis probe and three cats were positive with the T. foetus probe. All Trichomonas-positive cats were pure-bred and between 8 and 32 weeks of age. In one cat positive for T. foetus large amounts of parasites were found in the gut lumen and invading the intestinal mucosa. The species of the detected trichomonads were confirmed by polymerase chain reaction and nucleotide sequencing of a part of the 18S ribosomal RNA gene. In this study, the usefulness of CISH to detect intestinal trichomonads within feline tissue samples was shown. Additionally, the specific detection of P. hominis using CISH was established. Generally, it was shown that CISH is well suited for detection and differentiation of trichomonosis in retrospective studies using tissue samples. 相似文献