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1.
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
2.
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
3.
Polymerase chain reaction (PCR) was used to amplify the spacer regions between the 16S and 23S genes of rRNA genetic loci of Salmonella serovars for their rapid identification. These genetic loci revealed a significant level of polymorphism in length across the species/serovar lines. When the 16S-23S spacer region amplification products were subjected to agarose electrophoresis, the patterns observed could be used to distinguish all the serovars of Salmonella tested. Unique elements obtained in amplification products were mostly clustered at serovar level, although certain genus-specific patterns were also observed. On the basis of the results obtained, the amplification of 16S-23S ribosomal spacer region could suitably be used in a PCR-based identification method for Salmonella serovars. 相似文献
4.
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
5.
Jin J Xu D Narongwanichgarn W Goto Y Haga T Shinjo T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(3):273-276
The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene. 相似文献
6.
Conventional serological methods for the identification of canine mycoplasma isolates depend on the availability of a panel of species-specific diagnostic antisera and are not always reliable in terms of specificity. To enable simultaneous identification of field isolates, PCR-RFLP analysis of the 16S-23S rRNA intergenic spacer region was used to characterize the type strains of the 12 currently described canine mycoplasmas of the Genus Mycoplasma which represent the "classic" non-hemotropic species. The use of 16S-23S rDNA PCR in the first step of this analysis revealed specific size differences of amplicons which allowed to classify these 12 canine Mycoplasma species into three groups. Depending on the length of the amplicon, subsequent RFLP analysis of PCR products using two restriction endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding patterns. For further evaluation of the 16S-23S rDNA PCR-RFLP assay system as identification and differentiation tool, a total of 262 field isolates collected from the canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated with traditional serological test results. The remaining 11 isolates had an RFLP pattern distinct from the type strains included in this study and were identified by 16S rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay established in this study enabled a rapid, accurate and easily performed identification and differentiation of all 12 currently described non-hemotropic canine Mycoplasma species. 相似文献
7.
Nasal lavage fluid was collected from 155 tortoises, mostly Testudo spp., that were kept as companion animals and suffered from nasal discharge. Examination for chlamydial DNA by PCR assays targeting the ompA, ompB, and groESL genes, as well as the 16S rRNA signature region and the 16S-23S intergenic spacer, respectively, revealed 16 (10.3%) positive animals. Sequence analysis of PCR products indicated high homology to the family Chlamydiaceae. Phylogenetic trees constructed from partial sequences of the ompA and 16S rRNA genes showed that the present samples clustered outside the nine species of Chlamydia and Chlamydophila. Sequences of the nearest relative, Chlamydophila pecorum, were still clearly distinct from those of the positive tortoise samples. This suggests that the tortoises had been infected by Chlamydia-like agents, the taxonomic identity and pathogenic importance of which has yet to be established. 相似文献
8.
Hassan AA Khan IU Lammler C 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2003,50(4):161-165
Streptococcus dysgalactiae serogroup C, G and L strains were investigated by polymerase chain reaction (PCR) using oligonucleotide primers designed according to species-specific parts of the 16S-23S rDNA intergenic spacer region. The oligonucleotide primers with specificity for the 16S-23S rDNA intergenic spacer region allowed a correct identification of all S. dysgalactiae serogroups C, G and L strains investigated. No cross-reactivities could be observed with any of the control strains indicating the usefulness of PCR-technology to identify the serologically heterogeneous species S. dysgalactiae. 相似文献
9.
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
10.
2010年,辽宁铁岭某种鸡孵化场出现孵化率低、死淘率高的现象。通过对送检病死鸡胚进行病原检测,最终检测出5株革兰阴性杆菌和3种病毒,其中禽波氏杆菌和奇异变形杆菌的检出率分别为76．44％、61.36％,两者混合感染率达51．83％,而其他病原菌（大肠杆菌、沙门菌、绿脓杆菌）和病毒（AI,V、REV、CIAV）的检出率均较低。采用1对根据病原菌23SrRNA基因的共同保守序列设计的引物,以及根据临床常见致病菌16s～23SrRNA基因间隔序列（ISR）两端的16S及23SrRNA保守序列而设计的通用引物分别对分离菌进行PCR扩增。并分别测定所得片段的DNA序列。结果显示,所得DNA片段分别与GenBank中收录的登录号为HM545299的禽波氏杆菌和登录号为AY993943的奇异变形杆菌的相应核苷酸序列同源性达99．5％和98．7％。本研究最终确定禽波氏杆菌和奇异变形杆菌的混合感染是引起该场疫情发生的主要原因。 相似文献
11.
Jin J Narongwanichgarn W Xu D Goto Y Haga T Shinjo T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(3):285-287
The 16S-23S rRNA intergenic spacer regions (ISRs) of five strains of "Fusobacterium pseudonecrophorum" which had been proposed as a new species, were compared with those of F. varium ATCC 8501T. All the strains of "F. pseudonecrophorum" exhibited of sequence similarities of 97.7% to 100% to the strain of F. varium in their 16S-23S rRNA ISR sequences. This indicates that the strains of "F. pseudonecrophorum" and the type strain of F. varium are identical at the species level. 相似文献
12.
13.
Hassan AA Akineden O Lämmler C Huber-Schlenstedt R 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(5):257-259
In the present study three phenotypically CAMP-negative Streptococcus agalactiae, isolated from three cows with mastitis, were characterized by molecular analysis. An identification of the S. agalactiae was performed by conventional methods and by PCR amplification of species specific parts of the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. In addition all three phenotypically CAMP-negative isolates harboured a normal sized CAMP-factor encoding cfb gene indicating a reduced expression of CAMP-factor or a gene defect elsewhere along the pathway of expression. The clonal identity of the three isolates could be demonstrated by macrorestriction analysis of their chromosomal DNA. 相似文献
14.
Fournier C Kuhnert P Frey J Miserez R Kirchhofer M Kaufmann T Steiner A Graber HU 《Research in veterinary science》2008,85(3):439-448
Based on our clinical experience on bovine mastitis, we hypothesized that subtypes of Staphylococcus aureus (S. aureus) exist which differ in their contagious and pathogenic properties. In order to investigate this hypothesis, we analyzed strains of S. aureus isolated from spontaneous intramammary infection (IMI) with their virulence gene patterns and genotypes obtained by PCR amplification of the 16S-23S rRNA intergenic spacer (RS-PCR). The genotypes were then associated with epidemiological and clinical data including 26 herds. The results demonstrated a high association between genotypes and virulence gene patterns as well as between epidemiological and pathogenic properties of S. aureus. In particular, genotype B was related to high contagiosity and increased pathogenicity whereas the other types (C, OG) were found with infection of single cows. Because of the high clinical relevance, our results indicate the need to subtype the IMI-associated strains of S. aureus in the future. 相似文献
15.
Streptococcus dysgalactiae serogroup C, G and L strains were investigated by polymerase chain reaction (PCR) using oligonucleotide primers designed according to species‐specific parts of the 16S–23S rDNA intergenic spacer region. The oligonucleotide primers with specificity for the 16S–23S rDNA intergenic spacer region allowed a correct identification of all S. dysgalactiae serogroups C, G and L strains investigated. No cross‐reactivities could be observed with any of the control strains indicating the usefulness of PCR‐technology to identify the serologically heterogeneous species S. dysgalactiae. 相似文献
16.
Estuningsih S Soedarmanto I Fink K Lämmler C Wibawan IW 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(4):185-187
All 83 bacterial strains isolated from seven farms in three areas of the island of Java in Indonesia investigated in the present study could be identified as Streptococcus agalactiae. Identification was performed by cultural, biochemical and serological properties and by polymerase chain reaction amplification of species-specific parts of the gene encoding the 16S rRNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor (cfb) gene. All isolates were unpigmented. almost all of the isolates had the serotype pattern II/X. Despite these similarities a macrorestriction analysis of the chromosomal DNA of the bacteria revealed no significant homologies of the DNA-fingerprints of the S. agalactiae from the various areas. This last finding might possibly indicate that a single ancestral unpigmented serotype II/X S. agalactiae clone was responsible for the mastitis situation on Java and had evolved separately in the various farms and regions. 相似文献
17.
Barcellos DE de Uzeda M Ikuta N Lunge VR Fonseca AS Kader II Duhamel GE 《Veterinary microbiology》2000,75(2):189-198
The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli. 相似文献
18.
根据GenBank中乳酸杆菌16 S～23 S rRNA基因间沉默区序列设计引物,进一步鏊定猪源乳酸杆菌分离株HZ521;并通过体外试验,以嗜酸乳酸杆菌ATCC 4356为参考菌株,探讨HZ521株的益生素作用.部分16 S～23 S rRNA基因序列同源性分析结果表明,该分离株属于瑞士乳酸杆菌.HZ521株具有较强的产酸能力,能耐受强酸,对Hela细胞的黏附率为87.2%,显著高于ATCC 4356株(P<0.01).表明瑞士乳酸杆菌HZ521株具有益生素特性,可作为肠道益生素的候选菌株. 相似文献
19.
Nasal lavage fluid was collected from 155 tortoises, mostly Testudo spp., that were kept as companion animals and suffered from nasal discharge. Examination for chlamydial DNA by PCR assays targeting the ompA, ompB, and groESL genes, as well as the 16S rRNA signature region and the 16S-23S intergenic spacer, respectively, revealed 16 (10.3%) positive animals. Sequence analysis of PCR products indicated high homology to the family Chlamydiaceae. Phylogenetic trees constructed from partial sequences of the ompA and 16S rRNA genes showed that the present samples clustered outside the nine species of Chlamydia and Chlamydophila. Sequences of the nearest relative, Chlamydophila pecorum, were still clearly distinct from those of the positive tortoise samples. This suggests that the tortoises had been infected by Chlamydia-like agents, the taxonomic identity and pathogenic importance of which has yet to be established. 相似文献
20.
In a previous study we described the use of a new set of PCR primers (CpsiA/CpsiB) specific of the conserved pmp-family genes of Chlamydophila abortus as an efficient tool for the detection of these bacteria in ruminants including also preliminary results on avian strains. In this work, the use of this set of primers was extended to representative strains of the six major avian serovars (serovars A-F) and to field isolates of C. psittaci. For all the studied representative strains, using purified genomic DNA as a template, CpsiA/CpsiB primers allowed, as observed for C. abortus, a minimal 10-fold PCR signal increase compared to the one observed with ompA specific primers. In comparison to primers targeting the 16S-23S rRNA intergenic spacer, similar or increased sensitivity was observed depending on the strain. All the field isolates were amplified with CpsiA/CpsiB primers. On clinical samples, our primers are the best among those tested for detection of C. psittaci by simple conventional PCR. RFLP experiments performed using PCR fragments amplified with the CpsiA/CpsiB primers gave promising results demonstrating that these primers may provide an interesting tool for molecular typing when the bacterium cannot be grown from pathological samples. 相似文献