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1.
沙门菌是重要的人畜共患病原菌,血清型很多。不同种类、不同日龄的家禽均可感染,是严重危害养禽业的病原之一。本文通过血清学、分子生物学方法对上海部分鸡场的沙门菌进行了检测,与传统检测方法相比,缩短了检测时间,提高了检测的特异性和敏感性。实验结果发现禽伤寒/鸡白痢沙门菌、鸡肠炎沙门菌血清学为阳性的,经多重PCR鉴定,发现绝大部分不是禽源性沙门菌,由此可见,沙门菌多重PCR检测为临床诊断提供了一种快速、敏感和特异性高的诊断方法。 相似文献
2.
二温式多重PCR同时检测鸡的6种呼吸道病病原方法的建立 总被引：8，自引：1，他引：7
设计了6对分别针对鸡传染性支气管炎病毒（IBV），禽流感病毒（AVI）、鸡毒素霉形体（MG）、鸡传染性喉气管炎病毒（ILTV）、新城疫病毒（NDV）和滑液霉形体（MS）的特异性引物，根据反转录滞酶链反应（RT－PCR）和多重PCR原则，优化建立了一次PCR反应同时检测6种禽呼吸道病原的二温式多重PCR。该方法可同时扩增出6条区段大小与设计相符符的特异性片段，即IBV 1720bp,AIV 1050bp,ILTV647bp,NDV310bp,MS207bp，对人工感染鸡临床样品检测结果证明，该方法可用于临床诊断。 相似文献
3.
为有效确定引起鸡痛风的病原，本试验建立了同时检测鸡星状病毒(CAstV)、禽肾炎病毒(ANV)和鸡传染性支气管炎病毒(IBV)的多重PCR方法。针对IBV的N基因、CAstV和ANV的ORF-1b基因分别设计了3对特异性引物，通过优化退火温度等反应条件建立了多重PCR检测方法，并评估了该方法的特异性和敏感性，而后对临床痛风样本进行了检测。结果显示，多重PCR方法对3种病毒扩增产物的大小分别为1 600 bp(IBV)、794 bp(ANV)和350 bp(CAstV);该多重PCR检测禽马立克氏病毒、血清4型禽腺病毒、减蛋综合征病毒和J亚型禽白血病病毒均为阴性；病毒最低检测限IBV为1.96×10~2 copies/μL,ANV为2.10×10~2 copies/μL,CAstV为1.33×10~5copies/μL;多重PCR对临床病料的检测结果与单项PCR的检测结果一致。结果表明，本试验建立的多重PCR检测方法具有较好的特异性、敏感性和可靠性，为临床鸡痛风的诊断提供了准确、有效的技术手段。 相似文献
4.
1例鸡白痢沙门氏菌的分离鉴定与耐药性分析 总被引：1，自引：1，他引：0
为查明河南省新乡市某规模化蛋鸡场疑似沙门氏菌感染病例的病原，进而制定合理的治疗方案，本研究无菌采集疑似病雏鸡肠管样品，用革兰氏染色、培养特性观察、生化试验、PCR方法对其进行确诊，并进行病原回归试验，采用药敏纸片琼脂扩散法（K-B法）分析分离菌对19种常见抗生素的耐药性。结果显示，本试验成功分离了一株革兰氏阴性短杆菌，该分离菌符合鸡白痢沙门氏菌的培养特性和生化特性；PCR成功扩出invA基因，通过与GenBank数据库比对分析确定该细菌为鸡白痢沙门氏菌；用分离细菌株感染SPF鸡能复制出与自然感染一致的病例，说明鸡白痢沙门氏菌是造成本养鸡场雏鸡发病的主要病原；分离株具有较强的致病性和多重耐药性，对阿米卡星、苯唑青霉素、青霉素耐药，对复达欣、氨苄青霉素、头孢曲松等药物敏感，将敏感抗生素用于临床治疗效果明显。结合以上结果，本研究提出该病的具体防治措施，并取得了较好的效果，为鸡白痢沙门氏菌的分离鉴定及防治提供了参考，对鸡白痢沙门氏菌病病原的早期诊断和治疗具有重要临床意义。 相似文献
5.
应用多重套式PCR检测鸡毒支原体和鸡滑液囊支原体 总被引：2，自引：0，他引：2
根据已发表的鸡毒和鸡滑液支原体血凝素基因序列pMGA和vlhA各设计两对引物,建立鉴别诊断两种支原体的多重套式PCR方法,对其进行温度条件、Ⅱ步模板浓度优化及特异性、敏感性实验。该方法在两步PCR后能特异性地扩增出MG(408 bp)和MS(688 bp)两个目的片段。应用于临床样品检测,与支原体分离、SPA检测比较结果PCR灵敏度高于病原分离。 相似文献
6.
《黑龙江畜牧兽医》2017,(20)
根椐Gen Bank中已发表的白色念珠菌(CA)、传染性支气管炎病毒(IBV)、新城疫病毒(NDV)基因序列设计并合成三种病原的特异性检测引物,通过三重PCR反应条件的优化,建立了检测三种病原的PCR检测方法。该方法对同一样品中的CA、IBV和NDV核酸模板可同时扩增出231 bp、417 bp和747 bp的特异性片段,检测光滑念珠菌(CG)、克柔念珠菌(CK)、热带念珠菌(CT)、H9亚型禽流感病毒(AIV H9)、鸡传染性法氏囊病毒(IBDV)、鸡传染性喉气管炎病毒(ILTV)、减蛋综合症病毒(EDSV)等7种禽病病原均为阴性,该方法对CA DNA、IBV c DNA、NDV c DNA的检测最低限度分别为25.0,33.5,36.5 pg,临床样品检测结果表明三重PCR检测方法可以用于这三种病原单独和混合感染的检测和鉴别诊断。说明所建立的三重PCR检测方法具有很好的特异性、敏感性、重复性和临床适用性,为三种病原单独或混合感染的检测和鉴别诊断提供了一种操作简单、检测快速的分子生物学诊断方法。 相似文献
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多重聚合酶链反应快速检测鉴别鸡毒支原体强毒株和弱毒疫苗株的研究 总被引：8，自引：0，他引：8
根据鸡毒支原体强毒株和弱毒疫苗株基因组的结构特点，设计合成了二对引物XZ1，XZ2和XZ45、XZ46，建立了一种同时检测鉴别MG野毒株和弱毒疫苗株的多重PCR技术。试验结果表明，用这两对引物对MG强毒株和弱毒疫苗侏进行多重PCR，强毒株只扩增出732bp一条带，而弱毒疫苗株则可同时扩增出732bp、524bp二条带，而对其他种类鸡支原体和其它禽病病原的扩增不出现任何条带，结果均为阴性；敏感性测定结果表明，该多重PCR最低能检出1Pg的MG强毒株和弱毒疫苗株的DNA模板。 相似文献
9.
猪群中7种高发传染病多重PCR诊断方法的建立 总被引：4，自引：0，他引：4
[目的]建立猪瘟、蓝耳病(又称猪繁殖与呼吸综合征)、圆环病毒2型感染、猪链球菌病、副猪嗜血杆菌病、猪多杀性巴氏杆菌感染和猪支原体感染7种猪病的多重PCR诊断方法.[方法]根据GenBank发表的核苷酸序列设计了分别针对猪瘟、蓝耳病、圆环病毒2型感染、猪链球菌病、副猪嗜血杆菌病、猪多杀性巴氏杆菌感染和猪支原体感染的2套多重PCR特异性引物,优化多重PCR反应条件后进行敏感性与特异性评价.[结果]建立的2套多重PCR诊断方法对其他常见猪病痛原的基因组无特异性扩增,检测病原基因组最低浓度可达到pg级.[结论]该研究建立的2套多重PCR诊断方法可以用于猪群中上述7种猪病痛原的单一感染或混合感染的鉴别诊断. 相似文献
10.
PCR检测鸡毒霉形体和鸡滑液囊霉形体的研究 总被引：3，自引：0，他引：3
根据禽霉形体16S rRNA的基因序列设计，合成了1对引物，用这对引物对鸡霉素形体（Mycoplasma gallisepticum,MG)和鸡滑液囊霉形体（Mycoplasma synoviae,MS)菌株DNA进行PCR扩增，得到了与预期大小相一致的580bp的PCR产物，而这对引物对其他畜病病原DNA或RNA模板的扩增结果为阴性，PCR方法对MG和MS的最小检出量分别为2pg和3pg，应用已建立的PCR方法检测MG人工感染样品和临床样品，从人工感染样品中均检测到MG，临床样品的MG阳性检出率为10．25％，高于常规分离培养的阳性检出率。 相似文献
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Development and application of a multiplex polymerase chain reaction for avian respiratory agents 总被引：14，自引：0，他引：14
A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group. 相似文献
13.
A concurrent infection of chickens with infectious laryngotracheitis virus (ILTV), a herpesvirus, and fowlpox virus (FWPV), an avipoxvirus, is described. Two techniques, an immunohistochemistry (IHC) technique and a multiplex polymerase chain reaction (PCR), were used to examine 11 tissue samples from chickens clinically diagnosed as FWPV-infected, but only IHC was used to examine six tissue-paraffin blocks prepared from turkeys suspected of having FWPV infection. By multiplex PCR, both FWPV and ILTV were detected from three chicken samples (FI-90, FI-93, and FI-94); both FWPV and ILTV were detected from only two samples (FI-93 and FI-94) by IHC. All chicken samples were positive for FWPV by both PCR and IHC. Viral DNA from these samples was further confirmed by restriction enzyme analysis. When turkey samples were analyzed by the double-stain IHC, all six samples showed the presence of FWPV antigens, but no ILTV antigens. The double IHC technique, using monoclonal antibodies against FWPV and ILTV, was successful in simultaneous demonstration of specific FWPV and ILTV antigens colocalized in infected tissue samples as well as within individual cells. This paper emphasizes the importance of reliable tests that detect specifically the presence of ILTV and FWPV in infected tissue samples. The multiplex PCR assay holds potential to be versatile, rapid, and more sensitive (100%) than IHC (67%) for the simultaneous detection of two different avian viruses. Furthermore, the presence of mixed infection should always be kept in mind in the virologic analysis of respiratory sickness of poultry. 相似文献
14.
Simultaneous detection of three porcine viruses by multiplex PCR 总被引：2，自引：0，他引：2
Specific oligonucleotide primers were selected and combined in a multiplex arrangement, in order to detect simultaneously three economically important porcine viruses by polymerase chain reaction (PCR). The pathogen panel was comprised of viruses that cause reproductive failure in infected herds: Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV). In order to reduce the time required for the detection of the pathogens, the assay was optimised to a RapidCycler PCR instrument. The multiplex PCR assay was shown to be specific, sensitive and rapid, because the results were read in less than 60 min after sample preparation. Due to its speed, efficiency and sensitivity, the described rapid multiplex PCR assay serves as a useful novel tool in the veterinary diagnostic laboratories for the quick and complex detection of these important porcine pathogens. 相似文献
15.
S. Hutton J. Bettridge R. Christley T. Habte K. Ganapathy 《Tropical animal health and production》2017,49(2):317-322
A survey was conducted into respiratory infectious diseases of poultry on a chicken breeder farm run by the Ethiopian Institute of Agricultural Research (EIAR), located in Debre Zeit, Ethiopia. Oropharyngeal swabs were collected from 117 randomly selected birds, and blood was taken from a subset of 73 of these birds. A combination of serological and molecular methods was used for detection of pathogens. For the first time in Ethiopia, we report the detection of variant infectious bronchitis virus (793B genotype), avian metapneumovirus subtype B and Mycoplasma synoviae in poultry. Mycoplasma gallisepticum was also found to be present; however, infectious laryngotracheitis virus was not detected by PCR. Newcastle disease virus (NDV) was not detected by PCR, but variable levels of anti-NDV HI antibody titres shows possible exposure to virulent strains or poor vaccine take, or both. For the burgeoning-intensive industry in Ethiopia, this study highlights several circulating infectious respiratory pathogens that can impact on poultry welfare and productivity. 相似文献
16.
应用多重RT-PCR反应(mRT-PCR)结合变性高效液相色谱(DHPLC)技术建立禽产品中禽流感病毒和新城疫病毒的快速检测方法。以禽流感病毒NP基因和新城疫病毒F基因各设计1对引物,经优化单相RT-PCR(sRT-PCR)反应体系,建立多重RT-PCR(mRT-PCR)同时检测禽流感和新城疫,其PCR扩增产物经DHPLC技术进行快速检测分析后,检测极限分别达10-0.5ELD50/0.1mL和10-1.5ELD50/0.1mL。与普通凝胶电泳比较,敏感性高2个数量级。特异性试验表明,mRT-PCR-DHPLC仅检测到禽流感病毒与新城疫病毒,而对其他5种禽类病原体和SPF鸡胚尿囊液均未检测到阳性吸收峰。所建立的方法检测不同亚型禽流感病毒29株、不同来源及不同毒力的新城疫病毒16株,结果与实时荧光定量PCR检测结果完全一致;与病原分离法同时检测人工感染鸡的组织脏器,两种方法检测禽流感的符合率为94.4%(34/36),检测新城疫的符合率为95.4%(21/22)。病原分离法的检出率虽高于DHPLC方法,但经统计学分析两者差异不显著,两种方法同时对27份进口鸡胗样品与90份棉拭子样品进行检测,阴性符合率为100%,可适用于进出口禽肉产品的检测。 相似文献
17.
Salmonellosis is a common food-borne illness in humans caused by Salmonella-contaminated poultry and their products. In hatcheries, 110 Salmonella isolates were identified, mostly from first enrichment, and few from delayed enrichment. The Salmonella prevalence in goose and duck hatcheries was higher when measured by four multiplex PCR methods than by traditional culture (73.8% vs. 44.35%, P < 0.05); 97.3% of 110 isolates were Salmonella Potsdam of serogroup C1 and other isolates were Salmonella Montevideo of C1 and Salmonella Albany of C2. Plasmid and pulsed field gel electrophoresis genetic analysis revealed that isolates from duck hatcheries were more diverse than those from goose hatcheries. In Salmonella Potsdam, host species-specific genotypes were observed in geese for genotypes 3, 4, and 5 and in ducks for genotypes 7, 8, and 9, suggesting that Salmonella Potsdam may evolve into goose- and duck-specific isolates. An examination of 1121 eggs found that only Salmonella Potsdam was identified in 1.8% (7/591) of eggs from chickens fed on the ground, not housed in cages, and in egg content (6/7) as well as eggshell membrane (1/7). In conclusion, Salmonella Potsdam may be a major Salmonella infection in waterfowl and chicken eggs. 相似文献
18.
为建立一种可准确、快速鉴定畜禽临床病例中3种常见革兰氏阴性杆菌的多重PCR检测方法,本试验针对大肠埃希菌23S rRNA基因、巴氏杆菌KMT基因和沙门氏菌invA基因分别设计合成了1对特异性引物,构建可同时快速鉴别大肠埃希菌、巴氏杆菌和沙门氏菌的多重PCR反应体系,并进行反应条件优化及方法性能评估。结果显示,所建立的方法最佳引物浓度分别为1.0、1.5和1.0 μmol/L,最佳退火温度为56 ℃。性能评价结果显示,所建立的多重PCR检测方法具有较好的特异性和敏感性,其中对沙门氏菌检测敏感性最高,为1.44 pg/μL。对70株革兰氏阴性临床分离细菌进行检测,结果表明,所建立的多重PCR检测方法能实现3种临床分离细菌的快速准确鉴定。本方法的建立为相关临床病例快速诊断及流行病学调查提供了有效的技术支持。 相似文献
19.
MA Guang-qiang LIU Li-juan LONG Dan-dan YUE Jun LI Yi WEN Ming CHENG Zhen-tao ZHOU Bi-jun 《中国畜牧兽医》2016,43(3):577-584
To establish a rapid and accurate multiplex PCR method for the clinical detection of three main Gram-negative bacilli from livestock and poultry in Guizhou province,three pairs of specific primers were designed and synthetized according to 23S rRNA gene of Escherichia coli(E.coli),KMT gene of Pasteurella and invA gene of Salmonella.In this study,A mutiplex PCR reaction system was established for detecting the three pathogens at the same time,and the reaction system was optimized and it's performance was evaluated.The results showed that the best concentration of primers was 1.0,1.5and 1.0 μmol/L,respectively,and the best annealing temperature was 56 ℃.The results of performance evaluation showed that the method had good specificity and sensitivity,the sensitivity of Salmonella could reach to 1.44 pg/μL,was the highest.70 Gram-negative clinical isolated strains were detected by the multiplex PCR and it was proofed that the method could identify the three pathogens rapidly and accurately,and it could provide effective technical for the rapid clinical diagnosis and epidemiological survey. 相似文献
20.
Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples. 总被引：2，自引：0，他引：2
I S Schrank M A Mores J L Costa A P Frazzon R Soncini A Schrank M H Vainstein S C Silva 《Veterinary microbiology》2001,82(1):45-53
To attempt the rapid detection of Salmonella enterica, we have coupled a culture procedure with PCR amplification of the genus-specific invE/invA genes. The method was applied to different kinds of samples from the poultry industry and evaluated by using hydrolyzed feather meal, meat meal, litter and viscera, all experimentally inoculated with a known number of Salmonella followed by cultivation in selenite--cystine broth prior to the PCR reaction. The expected 457bp specific DNA fragment could be amplified from dilutions containing as few as 5.7CFU, indicating that the PCR technique can be successfully coupled with culture in an enrichment broth to distinguish Salmonella species from other enteric bacteria present in samples from the poultry industry. Tetrathionate broth proved to be a much better enrichment media compared to selenite-cystine when the presence of Salmonella was evaluated by PCR in 1-day-old chicks experimentally infected with known numbers of Salmonella. Samples included cecal tonsils and viscera, collected at 48h and 7 days postinfection. The PCR technique was more sensitive in detecting infected animals than the standard microbiological procedure, which detected only 47% of all PCR positive samples. 相似文献