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1.
A chronic and infective disease occurred on the farmed Chinook salmon (Oncorhynchus tshaivytscha) in a farm in Huairou district of Beijing and it caused high accumulative mortality.The typical clinical symptoms indicated that it might be bacterial kidney disease (BKD).The purpose of this study was to confirm its pathogen.The typical clinical symptoms of sick fish,morphology observation of bacteria in kidney tissue smears and sequence analysis of bacteria DNA were conducted.A number of gram positive brevi bacteria were observed in kidney tissue print.16S rDNA sequence of K1 strain was more than 99% homology with that of Renibacterium salmoninarum and they were in the same cluster.383 and 320 bp fragment were amplified by PCR and nested PCR.The fragments were sequenced and analyzed.The results showed the fragment was shared 100% nucleotide identity with P57 surface protein gene of Renibacterium salmoninarum. It was indicated that these diseased fish were suffering from BKD caused by Renibacterium salmoninarum.This was the first report about Renibacterium salmoninarum detected in farmed fish in China.  相似文献   

2.
北京怀柔某养殖场的大鳞大麻哈鱼发生一种慢性,但累计死亡率很高的流行病,为研究大鳞大麻哈鱼感染病原菌的种类,本试验根据病鱼临床症状观察判断疑似感染鲑肾杆菌.试验对病鱼肾脏组织进行印片染色观察细菌形态特征,抽提病鱼的肝脏、肾脏组织DNA,扩增16S rDNA和鲑肾杆菌P57表面蛋白基因片段,测序并运用BLAST进行同源性比对.结果显示,病鱼肾脏组织印片在显微镜下可见大量形态一致的革兰氏阳性短杆菌;采用病鱼肝脏、肾脏组织扩增的16S rDNA基因构建的系统发育树显示与鲑肾杆菌聚为一支;通过PCR扩增出鲑肾杆菌P57表面蛋白基因DNA片段,测序结果与鲑肾杆菌标准株ATCC 33209完全一致,符合率为100%.结果表明病鱼感染鲑肾杆菌并引发细菌性肾病(BKD).这是中国首次在养殖鱼体内检出鲑肾杆菌.  相似文献   

3.
The highly neurotropic Borna Disease Virus (BDV), which belongs to the Mononegavirales order--Bornaviridae family--is generally detected using the RT-nested-PCR. If false positive results (often caused by laboratory contaminations) can be avoided, some false negative results which are mostly due to inhibitory effects of some reaction components and/or to sample preparation errors, can occur. Thus, in order to control the RT-PCR sample, an RNA internal standard molecule named "mimic" was constructed with the same primer recognition sites as the viral nucleic acids, flanking a heterologous DNA fragment of distinct molecular weight. Because of their different sizes, the mimic and viral PCR products can be easily discriminated by agarose gel electrophoresis. The co-amplification of both BDV and mimic RNA was performed on infected cells and on biological tissues such as the brain and blood, commonly known to contain PCR inhibitor components. After mimic sensitivity studies were achieved (2.5 fg of "p40 RNA mimic" and 0.25 fg of "p24 RNA mimic"), the competitive amplification reaction between both BDV and mimic RNA was performed on these tissues. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity of BDV detection by a higher sensitive method such as RT nested PCR. Moreover, these results confirmed the interest of an internal standard for BDV RNA detection in biological samples.  相似文献   

4.
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   

5.
A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish.  相似文献   

6.
低分子质量蛋白抗原Mtb8.4是一种非常重要的结核分支杆菌抗原,为了研制结核病核酸疫苗和进行结核病的诊断,分别将其构建到原核和真核载体中进行表达。以结核分支杆菌标准菌株H37Rv基因组DNA为模板,PCR扩增目的基因Mtb8.4,扩增产物酶切后分别克隆到真核表达载体pJW4303和原核表达载体pGEX-4T-1中,构成重组真核表达载体pJW-Mtb8.4和重组原核表达载体pGEX-Mtb8.4,用限制性内切酶消化,PCR扩增及DNA序列分析等多种方法鉴定;并将正确构建的原核表达载体转入E.coliBL21(DE3)plysS中,IPTG诱导表达。结果表明,重组真核表达载体pJW-Mtb8.4和重组原核表达载体pGEX-Mtb8.4构建成功。构建的真核重组质粒pJW-Mtb8.4即可作为结核病DNA疫苗。原核表达载体pGEX-Mtb8.4在BL21(DE3)plysS中成功表达,将表达蛋白进行纯化,作为保护性结核分支杆菌抗原以便检测DNA疫苗的免疫效果。  相似文献   

7.
A serologic subgroup of bovine leukemia virus (BLV) has not been identified, whereas genetic diversity among BLVs has been reported by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). To investigate the distribution of BLV provirus variants, 42 isolates from Argentina and Japan were examined by nested PCR for a segment of the env gene, followed by DNA sequencing. The nucleotide sequences were compared with other previously characterized BLV variants from different geographical areas (Belgium, France, Italy, North America, Australia, Japan and Argentina). The majority of analyzed segments had a tendency for nucleotide substitution without changing the amino acid. The constructed phylogenetic tree showed the relations and differences between proviruses and within each one. Most of the samples in Argentina formed one cluster. The samples in Japan, except one, also formed one cluster and some of them showed high homology with the isolates from Australia and the USA. Considering the sequence analysis of env PCR products of all Japanese and Argentine samples and comparing them with the other previously isolated sequences, the variation was up to 3.5% and was characterized geographically in each area.  相似文献   

8.
Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.  相似文献   

9.
Antigen B (AgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. E. granulosus AgB is a gene family of at least five gene loci (B1-B5), each one consisting of several minor variants. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and polymorphism of AgB1 in 99 isolates which the 43 were from cattle, 25 of sheep and 31 of human. All samples were analyzed with 12S rRNA-PCR for the strain detection and all of were identified as G1-G3 cluster (E. granulosus sensu stricto). The 16 human, 35 cattle and 25 sheep isolates were yielded the 102 bp band by PCR and these samples were tested by SSCP. As results of the SSCP, different band profiles were detected one each of cattle and human isolates while the other 74 isolates showed same band patterns. The DNA sequence analysis was performed for these two isolates and the other selected 4 isolates and polymorphism was confirmed.  相似文献   

10.
The aim of this study was to determine if fluorescent PCR could be used instead of nested PCR, for the detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in clinical specimens, to improve the sensitivity without increasing the risk for cross-contamination. We investigated and compared the sensitivity of single PCR, fluorescent PCR and nested PCR for the detection of IS900, an insertion sequence specific for M. paratuberculosis. A previously described extraction method for clinical specimens, based on xylene, was evaluated regarding its suitability for routine diagnostic work. The sensitivity of each PCR system was assessed by analysing a serial dilution of M. paratuberculosis DNA. To improve the reliability of the PCR and to facilitate the interpretation of the PCR results, a positive internal control molecule ("mimic") was developed and used for single and fluorescent PCR. In nested PCR, an existing mimic was used. The efficiency of recovering DNA of M. paratuberculosis from clinical specimens by the extraction method and detection of the organism by PCR was studied by analysing spiked ileum mucosa specimens. The final evaluation was performed on seventeen ileum mucosa specimens, previously found positive for M. paratuberculosis by bacterial culture. Twelve of the samples were positive by fluorescent PCR and nested PCR, and 10 samples were positive by single PCR. The use of mimics showed inhibition in specimens harbouring few M. paratuberculosis organisms, illustrating the effect of inhibitory substances in combination with small amounts of M. paratuberculosis DNA. We conclude that the extraction method was not adequate to recover small amounts of M. paratuberculosis and that inhibitory substances were still present in the processed specimens, but that the method is useful for identifying positive samples. Fluorescent PCR was a suitable alternative to both single PCR and nested PCR for the detection of M. paratuberculosis.  相似文献   

11.
The aim of this study was to find suitable and reliable tools for demonstrating Lawsonia intracellularis in routine clinical diagnosis. Firstly, a method to prepare tissue samples before a polymerase chain reaction (PCR) was evaluated in pigs submitted for necropsy. Secondly, seven different faecal preparation methods and four different DNA polymerases were tested in single or nested PCR, with co-amplification of a mimic molecule. Thirdly, in selected pigs submitted for necropsy, tissue and faecal samples were examined histopathologically and by PCR, and blood samples were analysed serologically. Detection of L. intracellularis in tissue preparations by PCR showed good specificity and correlated to lesions found at necropsy. The sensitivity in spiked tissue samples was 10(1)-10(2) mimic molecules per tube. In faecal samples, nested PCR on boiled lysate gave the best result with a sensitivity of 10(2)-10(3) mimic molecules per reaction tube. However, because of the time-consuming procedure and the increased risk for contamination, a commercially available kit was preferred for routine diagnoses, despite a somewhat lower detection rate in subclinically infected pigs. In a few cases, the serological results differed from those obtained by PCR and by necropsy but the reason for this is not clear. This study indicates that the best method for diagnosis of acute enteritis in growers is PCR on faecal or tissue samples. To determine the presence of the bacteria in a herd, serology or repeated faecal sampling for PCR from target animals, or both, should be used.  相似文献   

12.
Specificity of six previously published Mycobacterium avium subsp. paratuberculosis (MAP) genomic loci, including 10, 38, 56, 93, 251, and 252 were evaluated in this study. Target 251 which was identified as MAP-specific was further evaluated in 210 MAP isolates, 14 non-MAP mycobacterial species, 7 atypical mycobacterial isolates, and 9 other bacterial species using real-time PCR. A previously published IS900 primer and probe combination was used as a positive control along with a universal ribosomal DNA gene sequence (UVA) as an internal control to evaluate PCR inhibition. All MAP isolates were positive with IS900, 251, and UVA by real-time PCR. All non-MAP mycobacterial species except one atypical mycobacterial isolate and other bacterial species used in this study were negative for IS900. All of these species were negative for 251. The atypical mycobacterial isolate, positive for IS900 and UVA, was negative for 251. A combination of IS900 and 251 PCR is ideal for sensitive and specific confirmation of MAP isolates from conventional fecal cultures. This study also evaluated the specificity of 251 real-time PCR, on broth cultures from 50 known bovine fecal samples. Acid fast staining followed by IS900 and 251 real-time PCR can be used for accurate identification and confirmation of MAP from broth cultures.  相似文献   

13.
Moraxella ovis was historically the only coccoid Moraxella identified in cultures of ocular fluid from cattle with infectious bovine keratoconjunctivitis (IBK) and could be morphologically and biochemically differentiated from Moraxella bovis. Moraxella bovoculi sp. nov. is a recently characterized Moraxella isolated from ulcerated eyes of calves with IBK in northern California in 2002. Like Moraxella ovis, M. bovoculi sp. nov. is a gram-negative coccus/diplococcus. All 18 original isolates of M. bovoculi sp. nov. possessed phenylalanine deaminase (PADase) activity and could therefore be differentiated from M. ovis and M. bovis. During the characterization of 44 additional isolates of hemolytic gram-negative cocci that were cultured from ulcerated eyes of IBK-affected calves, 2 PADase-negative isolates were identified that could not be differentiated biochemically from M. ovis; however, the DNA sequence of the 16S-23S intergenic spacer region (ISR) of the isolates matched the 16S-23S ISR DNA sequence of M. bovoculi sp. nov. To facilitate the identification of PADase-negative moraxellae, a polymerase chain reaction (PCR) coupled with restriction enzyme digestion analysis of amplified DNA was developed. Amplification of the 16S-23S ISR followed by AfaI digestion of amplified DNA could differentiate M. bovoculi sp. nov. from M. ovis and other moraxellae. The DNA sequence analysis of the amplified 16S-23S ISR from the 42 PADase-positive isolates of hemolytic gram-negative cocci indicated that all were M. bovoculi sp. nov. and all possessed an AfaI site. A PCR coupled with restriction analysis of amplified DNA can aid in identifying M. bovoculi sp. nov.  相似文献   

14.
OBJECTIVE: To evaluate a rapid polymerase chain reaction (PCR) fingerprinting technique for discriminating among Pasteurella multocida isolates from laboratory rabbits. SAMPLE POPULATION: 33 P multocida isolates from rabbits with clinical pasteurellosis. PROCEDURE: PCR assays were conducted with 2 minisatellites (core sequence and modified core sequence of phage M13) and 2 microsatellites ([GTG]5 and [GACA]4). Each bacterium was assigned to a PCR type for each of the primers used. Boiled bacterial extracts and purified genomic DNA were compared by use of PCR assays for phage M13 and (GACA)4. Plasmids were isolated from each bacterium, and their influence on PCR fingerprint was determined, using boiled extracts as a DNA source. RESULTS: M13 core sequence and M13 modified core sequence yielded 5 and 8 PCR types, respectively. The microsatellites (GTG)5 and (GACA)4 yielded 4 and 9 PCR fingerprint types, respectively. Fingerprint patterns obtained by use of isolated DNA differed from those obtained by use of boiled extracts, although discrimination among P multocida isolates was similar. The presence or absence of plasmids did not affect PCR fingerprints. CONCLUSION: Single primer PCR fingerprinting with minisatellite and microsatellite primers is an efficient and reproducible method for the discrimination of P multocida isolates from rabbits and can be performed directly, using boiled bacterial extracts as a source of template, although more bands were obtained from pure genomic DNA.  相似文献   

15.
PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, few studies have been performed with field samples in order to evaluate the assay as a diagnostic tool. In this study, routine use of a species-specific PCR assay previously developed for the identification of Brucella cultures was assessed for the detection of Brucella DNA directly from the stomach contents of aborted sheep fetuses. The assay is based on the insertion sequence IS711 in the Brucella chromosome. In the study, during 3 successive lambing seasons (1998-1999, 1999-2000 and 2000-2001) 126 aborted fetus samples each from different flocks and locations were examined. Brucella strains were isolated from 39 (31%) of the samples and all of the strains were identified as Brucella melitensis by biochemical characteristics, agglutination with monospecific A and M sera and PCR. Thirty-seven of 39 B. melitensis isolates were biotyped as biotype 3, and 2 isolates as biotype 1. From 38 of 39 culture positive fetal stomach contents B. melitensis-specific DNA was detected by PCR. PCR was found negative in all of the culture negative samples. Compared with culture, sensitivity and specificity of PCR were determined as 97.4 and 100%, respectively. The results indicate that this PCR procedure has a potential for use in routine diagnosis of sheep brucellosis.  相似文献   

16.
Salmonella Enteritidis is a leading cause of gastroenteritis associated with consumption of contaminated poultry meat and eggs. Because pulsed-field gel electrophoresis (PFGE) has limited utility in distinguishing between clonal Salmonella Enteritidis isolates, random amplified polymorphic DNA (RAPD) PCR has been recommended as an alternative molecular fingerprinting tool. This study's objective was to determine whether increasing PCR stringency would improve the repeatability of RAPD DNA patterns based on assessment of target sites within the genome. An in silico PCR was performed to predict amplification products from an Salmonella Enteritidis genome sequence for three different RAPD primers (1247, 1283, and OPA4) and to determine whether any primer would be more likely to amplify variable regions within the genome. A comparison of within- and between-isolate similarities in RAPD patterns was performed using primer 1247, which was predicted by in silico analysis to yield a variable size range of amplicons. In order to reduce artifactual variability associated with the method, three different methods for template preparation were evaluated. All were found to provide comparable results with respect to the similarities observed with repeated analyses of the same Salmonella Enteritidis isolates (n = 18, P = 0.91). Although the median within-isolate similarity (76.0%) was significantly greater than the median between-isolate similarity (66.7%; P = 0.001), duplicate RAPD-PCR runs of the same Salmonella Enteritidis isolates produced DNA patterns that ranged in similarity between 61.5 and 100%. These results indicate that the repeatability of RAPD-PCR is insufficient to distinguish genetic differences among related and unrelated Salmonella Enteritidis isolates.  相似文献   

17.
The present study was designed to comparatively investigate 25 Staphylococcus aureus strains isolated from bovine subclinical mastitis. The S. aureus strains, obtained from six different farms at five locations in one region of Germany, were characterized by phenotypic and genotypic methods. The S. aureus could be identified and further characterized by their cultural, biochemical and hemolytic properties. To analyze the epidemiological relationship the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNA, by PCR amplification of the gene encoding the 16S-23S rRNA intergenic spacer, by PCR amplification of the gene encoding the IgG binding region and the X region of protein A and by amplifying, and subsequent, digestion of the gene encoding staphylococcal coagulase. The macrorestriction analysis revealed five DNA restriction patterns with DNA patterns I, III and IV occurring in three, four, and three different farms, respectively. In addition, clones with different DNA patterns could be found within one herd. The PCR products for the spacer DNA, the spa gene encoding the X region of protein A and the coa gene encoding coagulase corresponded mostly to the pattern observed by DNA fingerprinting. Amplification of the gene encoding the IgG binding region revealed sizes of 620 bp for 20 of the isolates and 280 bp for four isolates indicating, for the latter, a deletion of segments in this region. These findings show, that single, widely distributed clones seemed to be responsible for cases of bovine subclinical mastitis found in one region of Germany.  相似文献   

18.
6株副猪嗜血杆菌基因组DNA的PCR指纹图谱研究   总被引：1，自引：0，他引：1  
根据肠道菌基因闻重复一致序列，设计了一对特异性引物，采用ERIC-PCR和RAPD技术，研究了副猪嗜血杆菌6个分离菌株的指纹图谱和DNA多态性。结果表明，6个分离株的PCR指纹图谱与15个标准血清型指纹图谱相比较可分辨出4种血清型；6个分离株的RAPD研究结果均表现出多态性。有意义的是，6个菌株的多态性DNA片段也能明显将其分为4种类型的副猪嗜血杆菌，与特异性引物PCR结果相一致。该研究可作为流行病学调查和该菌的分子分型快速诊断方法的基础。  相似文献   

19.
A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.  相似文献   

20.
Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.  相似文献   

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