共查询到20条相似文献，搜索用时 15 毫秒
1.
The aim of this study was to monitor chlamydial shedding patterns in clinically affected koalas before, during and following treatment using quantitative real-time PCR. Swab samples were obtained from 14 koalas presented for treatment at the Australian Wildlife Hospital. Four of these animals were followed over a period of 8–9 weeks. Primers were designed based on the consensus signature sequence of the 16S rRNA chlamydial gene. Additional primers were designed based on the sequence of the koala beta-actin gene and used to normalize chlamydial values when comparing results from different swab samples. Chlamydial 16S rRNA gene copy number was highest in swab samples from clinically affected sites. Daily injections of chloramphenicol resulted in a marked and rapid reduction in the numbers of chlamydiae being shed from all sites. In general, chlamydial copy number was no longer detectable by the end of the 2nd week of treatment. No evidence of relapse of infection was detected at 2 weeks after the cessation of treatment. In contrast, topical chloramphenicol treatment of the eyes required a longer treatment period and had little effect on the shedding of chlamydiae from other sites of the body. Further studies are required to confirm the efficacy of a shorter treatment period. 相似文献
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Comparison of culture, peroxidase-antiperoxidase reaction, and serum latex agglutination methods for diagnosis of chlamydiosis in pet birds 总被引：1，自引：0，他引：1
F M Moore M C McMillan M L Petrak 《Journal of the American Veterinary Medical Association》1991,199(1):71-73
Comparison was made among results of cloacal specimen culture, and cloacal swab specimen (cytologic) peroxidase-antiperoxidase (PAP), serum latex agglutination (LA), and tissue PAP assays for diagnosis of chlamydiosis in 144 birds. Swab specimen PAP findings correlated poorly with LA results and failed to predict the LA test result in any bird. Only 1 cloacal swab specimen was regarded as PAP-positive and was from the cloaca of a bird from which chlamydiae were isolated in culture. The sensitivity of swab specimen PAP, compared with culture results, was 33%, whereas specificity was 94%. In this study, swab specimen PAP was a less sensitive test, compared with culture, than was reported in a previous study. The sensitivity of LA in identifying birds that were cloacal culture-positive was poor; true-positive results were not detected, compared with culture results. The specificity of the LA method was 93%, compared with culture results. Results of the tissue PAP method correlated with culture results in the 3 birds for which both tests were performed. 相似文献
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Comparison of one-step RT-PCR and a nested PCR for the detection of canine distemper virus in clinical samples 总被引：3，自引：0，他引：3
Shin YJ Cho KO Cho HS Kang SK Kim HJ Kim YH Park HS Park NY 《Australian veterinary journal》2004,82(1-2):83-86
OBJECTIVE: To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens. DESIGN: A nested PCR was developed, compared to a one-step RT-PCR and validated. PROCEDURE: Two sets of specific primers for a one-step RT-PCR and a nested PCR, targeting a 640 bp fragment and a 297 bp fragment, respectively, were selected from the highly conserved region of the nucleocapsid protein (NP) gene of CDV. The nested PCR and the one-step RT-PCR were used to amplify a part of the CDV NP gene of a CDV vaccinal strain and samples of urine, blood, nasal discharge and saliva from 29 dogs suspected of suffering CD. RESULTS: Both the one-step RT-PCR and the nested PCR reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected 640 bp fragment of the NP gene was detected in 11/22 (50.0%) blood, 10/20 (50.0%) urine, 5/25 (20.0%) saliva and 6/27 (22.2%) nasal swab samples by one-step RT-PCR, whereas the nested PCR amplified an expected 297 bp fragment of the NP gene in 18/22 (81.8%) blood, 15/20 (75.0%) urine, 14/25 (56%) saliva and 19/27 (70.3%) nasal swab samples. CONCLUSION: The nested PCR detected CDV in blood, urine, nasal swab and saliva more frequently than did the one-step RT-PCR. Therefore, this assay should be a useful aid to antemortem diagnosis of CDV infections in dogs. 相似文献
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为了对羊口疮病毒(ORFV)陕西分离株B2L基因进行克隆、序列分析及原核表达,根据GenBank已登录的ORFV(JN565694.1)B2L基因序列,设计合成一对特异性引物,应用PCR技术扩增ORFV B2L基因,并将目的基因连接到原核表达载体pET-28a中,成功构建重组质粒PET28a-B2L,转化大肠埃希菌BL21感受态细胞进行诱导表达,并进行SDS-PAGE和Western blot分析。结果表明,成功克隆了ORFV陕西分离株B2L全基因序列,核苷酸序列分析表明,陕西分离株B2L基因与国内外已报道的ORFV毒株核苷酸同源性超过97.3%,氨基酸同源性超过95.0%。重组菌经IPTG诱导后成功表达分子质量约为42ku的重组蛋白,该蛋白能与羊口疮阳性血清特异性结合,具有良好的反应原性。研究结果为ORFV的分子生物学特性研究提供资料,为进一步研制ORFV单克隆抗体及抗体检测ELISA试剂盒奠定了基础。 相似文献
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Salisch H Ryll M Leise R Neumann U 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2000,47(1):27-35
Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) are an interesting alternative to digoxigenin-labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the detection of DNA or amplificate. This study examined the questions whether a SNAP probe might not only save time, but also increase the sensitivity of another PCR-based DNA probe test using a digoxigenin probe. Amplificates obtained by multispecies polymerase chain reaction (PCR), with either purified genomic DNA or DNA extracted from tracheal swabs taken in chicken flocks, were detected by both methods. The results for the clinical specimens were compared to culture. Under stringent conditions, the specificity and sensitivity obtained with the SNAP probe were comparable to the results obtained with the DIG probe. The quantities 10 fg (SNAP probe) and 100 fg (DIG probe) of purified Mycoplasma synoviae DNA were detected after amplification, but more positive clinical specimens were detected with the DIG probe. Under non-stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives as confirmed by culture. Because cross-reaction with Mycoplasma gallisepticum and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non-stringent conditions, it was concluded that, to take the full advantage of SNAP probes, their use in combination with species-specific primer pairs is recommended. PCR as a method for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono-infection is likely by serological results, non-stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma. 相似文献
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探针检测鸭黄病毒的地高辛标记DNA的制备与应用 总被引：1，自引：0，他引：1
利用RT-PCR方法扩增鸭黄病毒的NS3基因406bp的特异性片段,回收并纯化PCR产物,用地高辛标记,制备核酸探针。特异性试验结果表明,该探针仅与鸭黄病毒的核酸特异性杂交,而与鸭瘟病毒、H9N2禽流感病毒、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒的核酸杂交均为阴性。敏感性试验表明,该探针对鸭黄病毒的RNA最低检出限量为100μg/L。对疑似黄病毒感染鸭的肝脏、肺脏、脾脏、输卵管、卵泡膜和泄殖腔棉拭子进行检测,以卵泡膜的检出率最高。该研究为鸭黄病毒感染的诊断和流行病学调查提供了一种可靠的方法。 相似文献
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Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene. 总被引：2，自引：0，他引：2
M Giacometti J Nicolet K E Johansson T Naglic M P Degiorgis J Frey 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1999,46(3):173-180
A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity). 相似文献
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本实验通过设计针对伪狂犬病病毒gE基因的引物和MGB(Minor groove binder oligodeoxynucleotide conjugate,MGB-ODN)TaqMan探针,结合ABI PE7700荧光定量PCR仪器系统,建立了一种能区别伪狂犬病病毒野毒株和gE-疫苗株的快速检测方法gE-MGB-TaqMan PCR。实验表明,该方法可检测出最低43拷贝的gE基因和10~4倍稀释的伪狂犬病病毒Fa株DNA,与微量血清中和结合MTT比色法相比,灵敏度和特异性一致,检测时间仅为后者的1/10,操作比后者更为简单。特异性和重复性试验表明:gE-MGB-TaqMan PCR特异性和重复性好。该方法以闭管的模式操作,减少了各步骤污染的可能性,整个检测少于3 h。 相似文献
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Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) are an interesting alternative to digoxigenin‐labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the detection of DNA or amplificate. This study examined the questions whether a SNAP probe might not only save time, but also increase the sensitivity of another PCR‐based DNA probe test using a digoxigenin probe. Amplificates obtained by multispecies polymerase chain reaction (PCR), with either purified genomic DNA or DNA extracted from tracheal swabs taken in chicken flocks, were detected by both methods. The results for the clinical specimens were compared to culture. Under stringent conditions, the specificity and sensitivity obtained with the SNAP probe were comparable to the results obtained with the DIG probe. The quantities 10 fg (SNAP probe) and 100 fg (DIG probe) of purified Mycoplasma synoviae DNA were detected after amplification, but more positive clinical specimens were detected with the DIG probe. Under non‐stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives as confirmed by culture. Because cross‐reaction with Mycoplasma gallisepticum and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non‐stringent conditions, it was concluded that, to take the full advantage of SNAP probes, their use in combination with species‐specific primer pairs is recommended. PCR as a method for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono‐infection is likely by serological results, non‐stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma. 相似文献
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实时荧光定量RT-PCR方法检测禽流感病毒 总被引：3，自引：0，他引：3
根据禽流感病毒(Avian influenza virus,AIV)M基因上的保守序列,合成引物和荧光标记探针,以阳性AIVM基因质粒为标准品做标准曲线,建立了荧光定量逆转录聚合酶链反应(RRT-PCR)检测方法。结果表明,本试验建立的标准曲线循环阈值(Ct值)与模板浓度具有良好的线性关系,相关系数为0.999,灵敏度约为5拷贝/μL,相当于5个AIV颗粒,对新城疫病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为AIV检测提供了一种特异、敏感、快速的定量检测方法。对500份临床泄殖腔棉拭样品的检测,其结果阳性?阴性数与经典病毒分离方法符合率分别为91.2%?99.4%。在AIV临床样品筛检、流行病学监测等方面显示良好的应用前景。 相似文献
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为了快速、准确地检测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome,PRRSV),本研究根据PRRSV基因序列设计特异性引物和探针,建立了一种可同时检测PRRSV经典毒株、高致病性变异毒株以及近几年中国新出现的NADC30-like毒株的TaqMan-MGB实时荧光定量方法,并对该方法的特异性、敏感性和重复性进行验证,同时用建立的实时荧光定量方法与常规PCR方法对临床收集的120份疑似PRRSV样品进行检测。结果表明,该方法特异性良好,对PRRSV的经典毒株、高致病性变异毒株及NADC30-like毒株均有良好的扩增,但对猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(RV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)的检测结果均为阴性,无交叉反应;模板浓度在101～108拷贝/μL范围内具有良好的线性关系,标准曲线结果显示其扩增的相关系数为0.9999,扩增效率为93%;敏感性高,约是常规PCR方法的100倍,最低可以检测到101拷贝/μL的模板;重复性好,批内和批间重复性试验变异系数分别为0.17%～0.90%和0.65%～2.34%;用本研究所建立的实时荧光定量检测方法对120份临床样品进行检测,PRRSV阳性检出率为59.2%(71/120),而常规PCR方法的PRRSV阳性检出率为44.2%(53/120)。该方法的建立为PRRSV的实验室诊断、流行病学调查,以及预防和控制中国PRRSV的流行提供了快速、准确的检测手段。 相似文献
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为了建立快速简便的fneB-LAMP检测法用于马腺疫病原马链球菌马亚种(S.equi)的检测,本试验采用恒温水浴进行LAMP扩增,建立可视化fneB-LAMP检测方法.结果显示:对大肠埃希菌、无乳链球菌、金黄色葡萄球、蜡状芽孢杆菌和克雷伯杆菌5种菌与标准S.equi菌株检测,只能够检测出标准阳性菌株;对新疆昭苏县3个马... 相似文献
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根据GPV H1株核苷酸序列，设计了扩增VP1-VP3基因非重叠序列的1对引物，对其结构蛋白VP1与VP3非重叠核苷酸序列进行PCR扩增，将PCR产物纯化、回收后制备出GPV VP1-VP3基因DIG标记核酸探针，其标记效率达到0．1pg／μl。特异性检测结果表明，该探针能与GPV不同毒株核酸发生特异性杂交，而与对照的DPV、GPMV等病毒的核酸杂交反应均为阴性；敏感性检测结果表明该探针对GPV的最低检出量为0．032ng。上述试验结果表明该探针可以用于GPV感染临床病料的检测。 相似文献
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根据GenBank中已经发表的B亚型禽偏肺病毒F基因的保守序列设计并合成1对引物,利用RT—PCR扩增出1条与目的片段大小一致的725bp基因片段,回收、纯化PCR产物,用地高辛标记,制备出地高辛标记的aMPV核酸探针。特异性检测结果表明,该探针能与aMPV核酸发生特异性杂交,而与H9N2亚型AIV、NDV、IBV、ORT和E．coil的核酸杂交反应均为阴性;敏感性检测结果表明,该探针对aMPV的最低检出量为5Pg。应用制备的探针对山东省不同地区的605份商品肉鸡和122份商品肉鸭进行了核酸探针检测,阳性检出率分别为36．59％和34．51％。本试验制备的aMPV地高辛探针特异性强、敏感性好,对样品的检测结果表明山东省部分地区的商品肉鸡、肉鸭中普遍存在aMPV感染。 相似文献
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Henning K Sachse K Kirschen P Böhmer J Strutzberg-Minder K Grossmann E 《Berliner und Münchener tier?rztliche Wochenschrift》2005,118(1-2):1-7
The evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against chlamydiae in pig sera is described. The most widely used serological test is the complement fixation test (CFT). The CFT has a lack of sensitivity and specificity because of low antibody titers and unspecific reactions. Eight conventionally raised pigs were exposed to a pathogenic strain of Chlamydia suis, four controls were mock infected. The immune responses was monitored by CFT and indirect ELISA. There was no agreement between CFT and ELISA data. These results were confirmed by a study with 191 sera from nine pig farms. As shown by ELISA and PCR chlamydiae are widespread in swine. 相似文献
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旨在提高对禽流感病毒（avian influenza virus，AIV）检测效率，及时发现疫病。本研究通过分析M基因以及H5、H7和H9亚型的HA基因序列保守区域，设计并合成了相关探针和引物，建立了禽流感病毒（AIV）四重荧光RT-PCR检测方法，该方法可在检测禽流感病毒（AIV）的同时，确定病原是否为H5、H7和H9亚型。结果显示，该检测方法耗时短、特异性好、检测下限达到10-5 ng·μL-1。采用该方法检测临床采集的13个活禽交易市场的384份禽咽喉和泄殖腔双拭子样品，经检测，其中有60份样品为流感病毒阳性，且全部是H9亚型，该结果与行业标准方法（NY/T 772—2013）检测结果一致，κ值为1（P<0.001）。本方法能实现对禽流感病毒及H5、H7和H9亚型的高通量快速检测，将在AIV快速检测中发挥重要作用。 相似文献
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为建立一种快速、准确的非洲猪瘟病毒(ASFV)分子检测方法,以2018年8月分离的SY18毒株P72基因序列(GenBank登录号:MH713612.1)为参考毒株,人工合成质粒标准品,命名为pcDNA3-P72,基于该毒株P72基因序列,在1627~1876 bp处设计一对特异性引物和TaqMan探针,优化反应体系及条件,本研究成功建立了基于P72基因的TaqMan探针qPCR方法,并与OIE的特异性引物和TaqMan探针进行灵敏度、稳定性和特异性比较.结果显示:本研究建立的qPCR检测方法,标准曲线线性关系良好,R^2值可达到0.9932,灵敏性最低能检测到10 copies/μL,与OIE引物扩增灵敏度相当,比普通PCR方法高100倍.该方法重复性良好,批内和批间变异系数均小于1%.这一方法为我国非洲猪瘟疫情的快速确诊提供了必要的分子诊断工具. 相似文献