共查询到20条相似文献，搜索用时 15 毫秒
1.
Y. Sasaki K. Yamamoto K. Amimoto A. Kojima Y. Ogikubo M. Norimatsu H. Ogata Y. Tamura 《Research in veterinary science》2001,71(3):227-229
Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens. 相似文献
2.
Sasaki Y Yamamoto K Kojima A Norimatsu M Tamura Y 《Research in veterinary science》2000,69(3):289-294
In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene. 相似文献
3.
Sasaki Y Yamamoto K Kojima A Tetsuka Y Norimatsu M Tamura Y 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2000,62(12):1275-1281
Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg. 相似文献
4.
二重PCR方法鉴别气肿疽梭菌和腐败梭菌 总被引：1，自引：1，他引：0
用包含16S～23S rDNA间隔区和23S rDNA的部分序列作为气肿疽梭菌特异性标志,以α毒素部分序列作为腐败梭菌特异性标志,建立了鉴别气肿疽梭菌和腐败梭菌的二重PCR方法。结果显示：气肿疽梭菌C54-1株扩增出大小为509 bp的条带,腐败梭菌C55-1株、C55-16株均扩增出大小为148 bp的条带,均与预期吻合;而产气荚膜梭菌C57-1株、C59-37株,肉毒梭菌C62-4株,诺维梭菌C61-4株均未扩增出任何条带。扩增产物的测序结果进一步证实了本方法的特异性。菌株的生物学特性试验结果也符合相应气肿疽梭菌和腐败梭菌的特点。本研究所建立的二重PCR方法可用于气肿疽梭菌和腐败梭菌的快速鉴定。 相似文献
5.
The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia. 相似文献
6.
Kojima A Uchida I Sekizaki T Sasaki Y Ogikubo Y Tamura Y 《Veterinary microbiology》2001,78(4):363-371
We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens. 相似文献
7.
Prévot V Tweepenninckx F Van Nerom E Linden A Content J Kimpe A 《Zoonoses and public health》2007,54(8):320-327
Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth. 相似文献
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9.
根据羊魏氏梭菌A、B、C、D、E型共有的α毒素基因,设计了1对引物,通过对PCR反应条件进行优化,建立了羊魏氏梭菌PCR检测方法。该方法扩增条带大小为255 bp,最低核酸检测量A型为0.39 ng/L、B型为0.62 ng/L、C型为0.52 ng/L、D型为0.87 ng/L、E型为0.92 ng/L,而对羊大肠杆菌、羊链球菌、金黄色葡萄球菌、羊多杀性巴氏杆菌的扩增结果均为阴性。应用该PCR方法对54份临床样本进行检测,PCR检测结果高于细菌学和生化检测结果。结果表明,该PCR方法具有很好的特异性和敏感性,可用于临床羊魏氏梭菌病的早期快速诊断。 相似文献
10.
Davidson I Borenshtain R Weisman Y 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(2):83-87
The study describes three polymerase chain reaction (PCR) systems for the CVI988 vaccine virus: the meq gene, the MDV BamHI-D/H 132 bp tandem repeat fragment and the MDV-gB gene. Whereas the PCR product of virulent MDV strains and of the CVI988 virus strain with the meq and the 132 bp primer sets differed for the two templates, the MDV-gB PCR products were similar. The sensitivity of the three PCRs was determined for the two templates: the CVI988 DNA was detected up to 2.48 plaque forming units, and a MDV-1 DNA, was amplified with the 132 bp primers up to the 10(-3) DNA dilution, and up to the 10(-2) with the MDV-gB and meq gene primers. As conventional detection for the CVI988 vaccine virus is by tissue culture, the aim was to analyse the feasibility of the molecular detection of the vaccine virus in the vaccinated chick. In two experimental trials employing specific pathogen free and commercial Lohmann chicks, respectively, the vaccine virus replicated to a limited extent; it was detected only in the spleen of up to 60% chicks at 2-4 weeks and in one chick at 3 weeks, respectively. The survey of three commercial Lohmann flocks, kept in biosecurity conditions, revealed the vaccine virus only in the spleen of 40% of 30-day-old chicks. The present study shows that CV1988 DNA is present in vaccinated chicks in a low quantity and it is difficult to detect directly from the chick, probably because vaccine viruses are latent in vivo. For an efficient detection it is pertinent to cultivate the vaccine virus on chicken embryo fibroblasts (CEF), as then the virus escapes the latent state, enters into the productive mode of replication, and a high viral copy number is produced. 相似文献
11.
F A Uzal K Belak E Rivera C A Robles R E Feinstein 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1992,39(8):595-598
A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chauvoei, C. septicum and C. perfringens types B and C remained unstained by the PAP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria. 相似文献
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13.
RT-PCR检测禽传染性支气管炎病毒 总被引：2，自引：0，他引：2
用２对已发表的引物和１对自行设计的引物对同一禽传染性支气管炎病毒（ＩＢＶ）Ｈ１２０株进行ＲＴ－ＰＣＲ,分别获得了Ｓ１基因上与引物设计相一致的１７２０、２２８、６０２ｂｐ的扩增片段。用自行设计的引物对７个毒株（Ｈ１２０、Ｈ５２、Ｍ４１、Ｃｏｎｎ、Ｇｒａｙ、Ｔ、Ｈｏｌｔｅ）和５个分离株（宜毒、上毒、云毒、ＨＫ、１１８）的含毒尿囊液或纯化病毒进行ＲＴ－ＰＣＲ,结果除Ｈｏｌｔｅ株和２个分离株（宜毒、云毒）外,其余均成功地扩增出６００ｂｐ的片段。用１．７、０．２、０．６ｋｂ３对引物对６个ＩＢＶ毒株和６个分离株的含毒尿囊液在相同和不同条件下进行ＲＴ－ＰＣＲ,结果３对引物分别扩增出３、５、９株ＩＢＶ,同时可将不同血清型的１２个ＩＢＶ株分成６种基因型。将ＩＢＶ分离株ＨＫ与标准株Ｍ４１经ＰＣＲ扩增、ＨａｅⅢ和Ｈｉｎｄ酶切、ＲＦＬＰ分析,表明属同一马萨诸塞血清型。３株ＩＢＶ（Ｈ１２０,ＨＫ,Ｍ４１）在鸡胚中繁殖,ＰＣＲ最早检出的时间为２０～２４ｈ。ＲＴ－ＰＣＲ提供了直接从尿囊液和感染鸡组织中快速检测病毒的新方法。 相似文献
14.
Abe N Kimata I Iseki M 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2003,65(1):29-33
Giardia has been detected in domestic dogs in Japan, but the genotype of isolates has remained unclear because identification has relied on conventional microscopy. Here we tried to identify the genotypes of four isolates from dogs in Japan by direct sequencing of the PCR amplified Giardia glutamate dehydrogenase (GDH) gene. The primer pair GDHF3 and GDHB5, targeting the GDH gene, was designed to prime a region of the GDH gene sequence conserved in the strains found to have the dog-specific genotype. The specific PCR product (approximately 220 bp), amplified with this primer pair, was only observed when Giardia DNA was used as the template. The sequences of the diagnostic fragments were identical among the isolates from dogs, and were differed by 15 bp or 1 bp from the strains, which were found to be the dog-specific genotypes, Assemblage C or D respectively. To verify the identity of the amplified DNA, a phylogenetic analysis was performed. Consequently, the sequence of the isolates from dogs clearly clustered with the strain found to be Assemblage D with neighbor-joining analyses. Therefore, all the isolates from dogs examined were identified as the dog-specific genotype, Assemblage D. In the present study, we revealed the genotype of Giardia isolates in Japan, and showed that direct sequencing of the PCR product amplified with the primer pair GDHF3 and GDHB5 was a useful tool for distinguishing between the zoonotic and dog-specific genotypes. 相似文献
15.
Identification of duck plague virus by polymerase chain reaction 总被引：33，自引：0，他引：33
A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague. 相似文献
16.
根据GenBank上登录的犬瘟热病毒（Canine distemper virus，CDV）基因组全序列，选择CDV强、弱毒株间有区别保守区设计了一对通用引物P1和P4，并在该对引物跨越区域的内部设计了CDV强毒株特异性引物P2及弱毒株特异性引物P3，用引物P1／P4进行RT—PCR，然后用引物P2／P3／P4进行复合套式PCR，建立了一种能区分CDV强、弱毒株的复合反转录-套式聚合酶链式反应（RT—nPCR）的鉴别诊断方法。应用该方法从CDV强、弱毒株的基因组中分别扩增出了大小为247bp和177bp的特异性片段，从两种病毒基因组混合物中扩增出了大小为247bp和177bp的两条特异性片段，与犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒、新城疫病毒的细胞培养物以及正常细胞对照组进行复合RT—nPCR扩增时均为阴性。对从黑龙江省和吉林省采集的20份疑似CDV病料进行的检测结果表明，有15份类似CDV强毒，5份类似CDV弱毒。本研究建立的复合RT—nPCR可以有效检测CDV感染，能够将强、弱毒株区分开，可用于临床快速检测、流行病学监测以及追踪疫苗免疫效果等。 相似文献
17.
Sreedevi C Hafeez M Kumar PA Rayulu VC Subramanyam KV Sudhakar K 《Tropical animal health and production》2012,44(1):95-99
Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers,
one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp,
respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not
yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection,
out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis
in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive
for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect
up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that
PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment
of suspected cases of porcine cysticercosis. 相似文献
18.
鹅细小病毒和番鸭细小病毒双重PCR检测方法的建立 总被引：1，自引：0，他引：1
根据GenBank上登录的鹅细小病毒(GPV)和番鸭细小病毒(MDPV)基因序列,分别设计合成针对GPV非结构蛋白(NS)和MDPV NS2-VP1基因片段的2对引物GPV U/L和MDPV U/L,将GPV和MDPV提取核酸混合后作为模板,优化PCR反应条件,建立了能同时检测这2种病毒的双重PCR。特异性试验结果显示,引物GPV U/L仅特异性扩增出GPV-GZ1和GPV-GZ2株730bp核酸片段,引物MDPVU/L仅特异性扩增出MDPV的624bp核酸片段,双重PCR扩增出长度分别为730bp和624bp的2条特异性片段,而扩增鸭瘟病毒(DPV)和鹅副黏病毒(GPMV)的核酸扩增结果均为阴性。敏感性试验结果显示,双重PCR能同时检测到14.4pg的GPV核酸和28.8pg的MDPV核酸。结果表明,建立的双重PCR可用于GPV和MDPV的鉴别诊断和联合检测。 相似文献
19.
The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline. 相似文献
20.
本研究旨在建立能同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV2)、坏死梭杆菌(Fn)和副猪嗜血杆菌(Hps)7种猪场常见高致死性流行病原的多重PCR检测方法。利用7种病原体的保守序列设计7对特异性引物,同时合成了Cy-5标记的通用引物。将通用引物分别连接到特异性引物的5′端形成7对特异性嵌合引物。优化反应条件,分别使用7组嵌合引物和通用引物混合,扩增7种病原的混合cDNA/DNA,验证其单重PCR的特异性。利用GeXP多重基因表达遗传分析系统,混合7种嵌合引物和通用引物,扩增单一病原的cDNA/DNA,验证其多重PCR特异性;将其他常见猪病病原的基因组作为干扰的阳性标本,利用7对混合嵌合引物和通用引物进行多重PCR分析,扩增加入了阳性标本的混合模板,验证其多重PCR的抗干扰能力。利用重组质粒和体外转录的RNA进行梯度稀释,确定GeXP多重检测体系的灵敏度。结果表明,7种不同引物分别进行GeXP单重及多重检测,均能检测出特异性目的片段的信号,无明显的干扰片段信号出现;GeXP多重检测抗干扰试验结果显示,在混入3种干扰病原模板后,依然可同时特异性检测出7种病原;GeXP多重检测灵敏度分析显示,在10~3拷贝/μL浓度条件下能检测到7种不同基因的特异性结果。本研究建立的同时检测7种猪场常见高致死性流行病原的GeXP检测方法具有高通量、高特异性和高灵敏度的特点,为快速诊断猪流行性疾病的交叉感染和混合感染提供了新型的检测方法。 相似文献