共查询到20条相似文献，搜索用时 406 毫秒
1.
Aihong Liu Guiquan Guan Pengfei Du Huitian Gou Jun Zhang Zhijie Liu Milin Ma Qiaoyun Ren Junlong Liu Jifei Yang Youquan Li Qinli Niu Qi Bai Hong Yin Jianxun Luo 《Veterinary parasitology》2013,191(1-2):15-22
The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species – T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61 °C or 63 °C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species – T. sergenti and T. sinensis, especially in endemic countries. 相似文献
2.
片形吸虫DNA随机扩增多态性分析 总被引：7，自引：1，他引：6
为区别从南京市江宁县采集的片形吸虫非典型形态虫体，应用随机扩增多态性DNA（RAPD）技术，对6株片形吸虫总DNA进行了扩增。结果，10条引物中有8条能产生扩增图谱，电泳图谱经聚类分析，与传统的分类结果一致，并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫，也有形态不典型的大片形吸虫。 相似文献
3.
Alasaad S Soriguer RC Abu-Madi M El Behairy A Jowers MJ Baños PD Píriz A Fickel J Zhu XQ 《Veterinary parasitology》2011,179(1-3):266-271
Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. 相似文献
4.
WANG Hua-jun LIU Yu JIANG Wei ZHAO Xue-li CAO Wei-wei CHEN Han-zhong YAN Ruo-qian 《中国畜牧兽医》2017,44(1):254-261
To establish a rapid, simple, sensitive and adapting field application assay for water buffalo Fasciola gigantica, the monoclonal antibodies of hybridoma cell strains 7D1 and 7D4 against excretory-secretary antigen(ES Ag) of Fasciola gigantica were prepared and purified. Meanwhile, the colloidal gold particle was prepared by the sodium cirrate reduction method, then the affinity purified 7D1 monoclone antibodies was labeled with the 30 nm colloidal gold particles. The 7D4 monoclonal antibodies and the goat anti-mouse IgG antibody using as secondary antibodies were coated on the surface of nitrocellulose filter(NC) membrane as the test line (T line) and control line (C line), respectively. The immune colloidal gold test strip was developed by optimizing the experiment conditions. The test results showed the prepared strip had a detecting prescribed minimum of 0.94 μg/mL of Fasciola gigantica excretory-secretary antigen, and it had no any cross reaction with the antigens of Eurytrema pancreaticum, Paramphistome, Chlamydia, Toxoplasma gondii and Trypanosoma evansi. Using the prepared strip to investigate the 334 fresh feces from buffaloes in Guangxi and the positive rate was 38.62%. These results indicated that this strip method was simple, rapid and easy determination, good specificity, high sensitivity, and adapted field application assay for Fasciola gigantica. 相似文献
5.
为建立一种快速、简便、适应现场应用的水牛大片形吸虫检测方法,本试验利用抗大片形吸虫ES抗原单克隆抗体杂交瘤细胞株7D1和7D4分别制备和纯化得到单克隆抗体7D1和7D4;采用柠檬酸三钠还原法制备颗粒大小为30 nm的胶体金,再用胶体金标记纯化单克隆抗体7D1,在硝酸纤维素膜的质控带和检测带处分别包被羊抗鼠IgG二抗和单克隆抗体7D4,经反应条件优化,组装成了大片形吸虫免疫检测试纸条。经测试该检测试纸条对ES抗原的检测下限为0.94 μg/mL,与胰扩盘吸虫、前后盘吸虫、支原体、弓形虫、伊氏锥虫均无交叉反应,特异性好。采集广西地区334份水牛新鲜粪样并用试纸条检测,阳性率为38.62%。结果表明,试纸条方法操作简单、快速、易于判定,特异性好,敏感性高,适合基层大面积普查和现场检测大片形吸虫。 相似文献
6.
Luo Y Sahin O Dai L Sippy R Wu Z Zhang Q 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(5):591-596
Loop-mediated isothermal amplification (LAMP) assay is a simple, rapid and specific detection method and has been used for detection and identification of different Campylobacter species. In this study, we develop a LAMP assay specific for detection of a particular clone (clone SA) of Campylobacter jejuni, associated with the vast majority of recent sheep abortions in the U.S. Using a set of specific primers for C. jejuni IA3902 (a clone SA isolate) and genomic DNA or boiled cell extract as template, the target DNA was amplified at 63 °C for 50 min in a water bath. A positive reaction was identified visually as white precipitate or fluorescence under UV, and confirmed by gel electrophoresis. Detection limit of the assay was comparable to that of conventional PCR. The LAMP was shown to be specific for detection of clone SA when tested on a number of C. jejuni strains of different genetic backgrounds. Applicability of the LAMP assay for specific detection of clone SA was demonstrated in animal tissues experimentally infected with IA3902 or genetically diverse C. jejuni strains. Since clone SA is the predominant cause of sheep abortions in the U.S. and is a zoonotic pathogen, the LAMP assay may be a valuable detection tool in future epidemiological studies. 相似文献
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Pleasance J Raadsma HW Estuningsih SE Widjajanti S Meeusen E Piedrafita D 《Veterinary parasitology》2011,178(3-4):264-272
In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes. 相似文献
9.
In order to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Torque teno sus virus type 2 (TTSuV2), two pairs of primers were designed according to the untranslated regions and the part of open reading frame 1 of TTSuV2.The LAMP system was optimized by adjusting the concentrations of some components and reaction conditions.The optimized amplification conditions of LAMP assay was at 64 ℃ for 90 min.The results showed the LAMP assay was specific for TTSuV2 detection, which could achieve a detection limit of 100 copies/μL viral nucleic acid, and no cross-reaction with TTSuV1, PCV2, CSFV, PRRSV and PBoV.In conclusion, this assay was a rapid, specific and sensitive detection technique which could provide a assistance for the rapid detection of TTSuV2. 相似文献
10.
为了能快速、特异的检测猪细环病毒2型(TTSuV2),本研究针对TTSuV2全基因序列的非编码区域和第1个开放性阅读框前端设计了2对引物,建立了TTSuV2的环介导等温扩增(LAMP)检测方法并对反应成分和条件进行了梯度摸索。试验结果显示,该LAMP检测方法最佳反应条件为64 ℃恒温90 min,可特异性检测TTSuV2,与猪细环病毒1型、猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒和猪博卡病毒无交叉反应,病毒最低检出限为100拷贝/μL。结果表明,建立的LAMP方法具有快速、特异且灵敏的特点,可在TTSuV2快速检测方面提供一定的技术支持。 相似文献
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W M Lotfy H N El-Morshedy M Abou El-Hoda M M El-Tawila E A Omar H F Farag 《Veterinary parasitology》2002,103(4):323-332
Reports on the species of Fasciola present in the Nile Delta, Egypt, appear controversial. Some authors reported the presence of both Fasciola gigantica and Fasciola hepatica, others reported F. gigantica only and mentioned that F. hepatica was found only in imported animals.This study was an attempt to identify the species of Fasciola flukes collected from locally bred animals. Morphologic, morphoanatomic, morphometric, and chemotaxonomic criteria of the fluke isolates were studied. Speciation based on morphologic and morphometric data was not decisive due to overlap in the values of most measurements. Morphoanatomic data proved the presence of both the species, and isoelectric focusing (IEF) of fluke soluble protein confirmed the presence of both F. gigantica and F. hepatica in Egypt. 相似文献
13.
本试验旨在培育大片形吸虫囊蚴,着重研究大片形吸虫在中间宿主体内的发育过程和临床病理变化.人工培养和孵化大片形吸虫虫卵,用孵化出来的大片形吸虫毛蚴感染中间宿主小土蜗螺,收集大片形吸虫囊蚴,再用囊蚴感染试验小鼠.结果显示,囊蚴经口感染试验小鼠后1周即可在肝脏找到虫体,幼虫可在小鼠体内发育生存7～8周,随着时间的推移和感染囊蚴数量不同,可给小鼠肝脏造成不同程度的病变,甚至造成死亡.剖检小鼠可见肝脏质地脆,颜色发黄,脾脏肿大等严重病理变化.因此,可利用小鼠作为大片形吸虫幼虫感染的试验动物,进行片形吸虫病的早期诊断、免疫预防和治疗等方面的研究. 相似文献
14.
为提高双芽巴贝斯虫(Babesia bigemina)检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的B.bigemina检测方法。根据GenBank上公布的Babesia bigemina细胞色素b(Cytochrome b,cyt b)基因序列,设计4条特异地识别B.bigemina的cyt b基因6个特殊区域的LAMP引物,优化反应体系和条件,在Bst DNA聚合酶的作用下,65 ℃反应60 min,加入SYBR Green Ⅰ后观察。结果表明,该LAMP检测方法特异性强,与牛巴贝斯虫(Babesia bovis)等DNA不发生交叉反应;敏感性高,对B.bigemina的cyt b基因最小检测值为0.085 fg/μL,是一般PCR方法的1000倍。该方法具有简单、快速、低成本的特点,可用于B.bigemina的基层现场快速检测。 相似文献
15.
Xie Z Tang Y Fan Q Liu J Pang Y Deng X Xie Z Peng Y Xie L Khan MI 《Avian diseases》2011,55(4):575-579
A loop-mediated isothermal amplification (LAMP) assay was optimized for the rapid detection of Group I avian adenoviruses. A set of six primers was designed from the DNA sequences of hexon genes from Group I avian adenovirus. The assay was performed in a water bath for 60 min at 63 C, and the amplification result was visualized by adding a fluorescence dye reagent or by inspecting the white sediment. The results showed that the LAMP assay could detect all 12 serotypes of Group I avian adenovirus and nine Guangxi Group I avian adenovirus isolates. This avian adenovirus Group I-specific LAMP assay could detect 238 copies of avian adenovirus. No cross-reactions were detected using the LAMP assay with avian adenoviruses type II and III or with other avian viruses. The ability of LAMP to detect Group I avian adenovirus isolates was further evaluated with 184 cloacal swab samples from poultry. In total, 72 out of 184 cloacal swab samples from poultry were identified as positive by LAMP, whereas 45 out of 184 were identified as positive by conventional PCR test. The Group I avian adenovirus specific LAMP results were further confirmed by real-time PCR. This specific LAMP method holds promise as a rapid and specific diagnostic assay for detection of samples from birds suspected of adenovirus infection. 相似文献
16.
应用环介导等温扩增技术快速检测单核细胞增生李斯特菌的研究 总被引：1，自引：0，他引：1
目的建立环介导等温扩增技术快速检测单核细胞增生李斯特菌。方法根据单核细胞增生李斯特菌(LM)hlyA基因序列中的保守区域,采用在线引物设计软件Primer Explorer4.0进行设计,获得一套特异性的环介导等温扩增(LAMP)引物,对单核细胞增生李斯特菌hlyA基因进行LAMP扩增,并与常规PCR方法进行比较。结果建立的LAMP方法能成功扩增出梯形条带,LAMP检测单核细胞增生李斯特菌纯培养物和人工染菌的灵敏度为5.44×102cfu/mL,而对照PCR检测的灵敏度为5.44×104cfu/mL。对10株细菌进行LAMP扩增,仅单核细胞增生李斯特菌得到阳性结果。从DNA提取到报告结果,耗时仅1h。结论 LAMP检测单核细胞增生李斯特菌灵敏度高,特异性强,耗时短,方法简便,有望发展成为快速检测食品中单核细胞增生李斯特菌的有效手段。 相似文献
17.
A loop-mediated isothermal amplification (LAMP) assay was developed for detection of Salmonlla in fecal samples of experimental monkeys. According to the specific sequences (fimY) of Salmonlla in GenBank, one set of primers was selected and the reaction condition was optimized. The results showed that the detection limit of LAMP method was 1.35×101 and 1.35×103 CFU/mL in Salmonella pure culture and clinical samples,respectively,which was the same as routine PCR. The amplification could complete in one hour, and the result could be distinguished by naked eyes. There was non-specific amplification of other pathogens. These results suggested that this LAMP assay was a simple and specific method for rapid detection of Salmonlla in fecal samples. 相似文献
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Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence 总被引：5，自引：0，他引：5
Isolates of Fasciola (Platyhelminthes: Trematoda: Digenea) from different host species and geographical locations in Mainland China were characterised genetically. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified from individual trematodes by polymerase chain reaction (PCR), and the representative amplicons were cloned and sequenced. The length of the ITS-2 sequences was 361-362bp for all Chinese Fasciola specimens sequenced. While there was no variation in length or composition of the ITS-2 sequences among multiple specimens from France, Sichuan and Guangxi, sequence difference of 1.7% (6/362) was detected between specimens from France and Sichuan, and those from Guangxi. Based on ITS-2 sequence data, it was concluded that the Fasciola from Sichuan represented Fasciola hepatica, the one from Guangxi represented Fasciola gigantica and the one from sheep from Heilongjiang may represent an "intermediate genotype", as its ITS-2 sequences were unique in that two different ITS-2 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is almost identical to that of F. gigantica in that nucleotides at five of the six polymorphic positions represent F. gigantica. This microheterogeneity is possibly due to sequence polymorphism among copies of the ITS-2 array within the same worm. Based on the sequence differences, a PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay was established for the unequivocal delineation of the Fasciola spp. from Mainland China using restriction endonuclease Hsp92II or RcaI. This assay should provide a valuable tool for the molecular identification and for studying the ecology and population genetic structures of Fasciola spp. from Mainland China and elsewhere. 相似文献
20.
Polymerase chain reaction (PCR) was used to detect Fasciola gigantica infection in the snail intermediate host. Fasciola specific primers amplified a 124 bp fragment in PCR when the genomic DNA isolated from F. gigantica infected Lymnaea auricularia snails was used as template. In addition to the 124 bp amplicon, a ladder of DNA fragments representing amplification of the 124 bp repetitive sequences was observed. Genomic DNA of the parasite was used as a positive control, which also gave an amplification of the 124 bp fragment. DNA isolated from non-infected snails was used as a negative control and no amplification of this sequence was observed. This technique is highly specific and sensitive and possesses fairly good prospects of its utility as an epidemiological tool for ascertaining the infectivity status in ubiquitous snail populations. 相似文献