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1.
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   

2.
Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.  相似文献   

3.
Four genetic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16s rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.  相似文献   

4.
鸡毒支原体PCR检测试剂盒的研制与应用   总被引：2，自引：0，他引：2  
根据基因库中鸡毒支原体 1 6SrRNA的序列研制PCR检测试剂盒 ,用于检测鸡毒支原体 (MG)。结果表明该MG PCR检测试剂盒对不同MG参考菌株和地方分离株均能特异性地扩增出 732bp条带 ,而对其他禽支原体和禽病病原体的扩增结果为阴性。该MG PCR试剂盒最低能检测出 1 0 0fg的MGDNA模板。保存期测定结果表明 ,该MG PCR试剂盒在 - 2 0℃条件下保存至 1 ,3 ,6和 9个月时 ,其敏感性无明显变化 ,仍能检测到 1 0 0fg至 1pg的MGDNA模板。保存至 1 2个月时其敏感度虽降低了 1个滴度 ,但仍能 1 0 0 %检出人工感染鸡的临床样品  相似文献   

5.
Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.  相似文献   

6.
通过PCR扩增分离保存的8株禽波氏杆菌、3株兔支气管败血波氏杆菌及1株猪支气管败血波氏杆菌菌株的23S rRNA基因的片段(710bp),分别克隆到载体pMD18-T后测序。利用BLAST工具进行同源性搜索;用DNAStar分析软件进行同源核苷酸序列的多重比较分析并构建禽波氏杆菌菌株的系统生物进化树。结果显示,8株禽波氏杆菌核苷酸序列之间同源性为99.2%～99.7%,与GenBank中收录的NC_010645株禽波氏杆菌核苷酸序列同源性为92.4%～92.5%,与兔支气管败血波氏杆菌和猪支气管败血波氏杆菌的核苷酸序列同源性均为98.5%～99.2%。国内分离的禽波氏杆菌菌株和支气管败血波氏杆菌菌株均与国外分离菌株在遗传基因上有一定的差异。结果表明,23S rRNA序列分析可以作为鉴定禽波氏杆菌的一种快速简便的方法。  相似文献   

7.
通过PCR反应扩增7株鸡奇异变形杆菌和1株兔奇异变形杆菌的23SrRNA基因片段(1045bp),经克隆测序,用DNA Star分析软件将所获得的序列与GenBank中收录的奇异变形杆菌、普通变形杆菌以及其他相近属的23S rRNA基因序列进行同源性比较,并由此构建奇异变形杆菌系统进化发生树。结果表示,本实验室保存的7株鸡奇异变形杆菌核苷酸序列与GenBank中收录的奇异变形杆菌核苷酸序列同源性为99.4%～99.8%,与兔奇异变形杆菌核苷酸序列同源性为98.8%～99.3%,与普通变形杆菌的核苷酸序列同源性为95.4%～96.2%,而与其他相近属同源性只有92.9%～93.4%。结果表明,23S rRNA基因序列分析可以作为鉴定奇异变形杆菌的一种快速、简便的方法。  相似文献   

8.
The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects.  相似文献   

9.
We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.  相似文献   

10.
根据鸡毒支原体(MG)和滑液囊支原体(MS)的16SrRNA基因序列，设计并合成了探针MG和MS共同目的基因，跨幅为580bP的一对引物。用这对引物对2个标准的MG和1个标准的Ms菌株DNA模板进行聚合酶锭反应(PcR)，结果得到了预期大小约580bP的PcR产物。回收纯化琼脂糖电泳凝肢中的MG扩增产物克隆至T载体中，DIG随机引物法合成核酸探针，斑点杂交试验对MG和MS均呈阳性，检测灵敏度分别为2ng和3ng，其它对照菌(毒)株为阴性。  相似文献   

11.
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   

12.
Mycoplasma felis is associated with conjunctivitis and respiratory disease in domestic cats. Currently no rapid diagnostic test is available for the detection of M. felis in clinical samples that does not rely on prior cultivation of the organism. The objective of this study was to determine whether a polymerase chain reaction (PCR) based upon the 16S/23S rRNA intergenic spacer sequence is suitable for the identification of M. felis directly in feline clinical samples. The high conservation between the 16S/23S rRNA intergenic spacers (IGS) of differing isolates of M. felis was established by sequence analysis and a PCR was developed to this region by comparison to IGS of other mycoplasmas. The PCR was found to be highly specific for M. felis and further PCR analysis on clinical samples showed the PCR to be highly sensitive and more rapid than the other methods of identification currently available.  相似文献   

13.
The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.  相似文献   

14.
甘肃地区牛源金黄色葡萄球菌分子鉴定及RAPD分型   总被引：1，自引：0，他引：1  
本研究目的是分离鉴定引起甘肃地区奶牛乳房炎的金黄色葡萄球菌,掌握其基因型情况。利用16S、23SrRNA保守序列PCR扩增对乳房炎奶样中的金黄色葡萄球菌进行鉴定,并进行RAPD基因分型。结果表明,310份奶样中共分离出金黄色葡萄球菌100株,RAPD结果显示这100株金黄色葡萄球菌均可得到清晰的RAPD指纹图谱,扩增产物在2～7条带之间,具有多种带型组成。通过聚类分析100株菌产生11个基因型,其中Ⅰ型4株,Ⅱ型4株,Ⅲ型10株,Ⅳ型13株,Ⅴ型7株,Ⅵ型24株,Ⅶ型16株,Ⅷ型6株,Ⅸ型4株,Ⅹ型10株,Ⅺ型2株。Ⅵ型为该地区的流行优势菌群,不同牛场各基因型菌株分布有明显差异。本研究说明牛场的环境与养殖条件对病原菌流行传播有明显的影响,这一结果对地区性奶牛乳房炎的防治提供了可靠的理论依据。  相似文献   

15.
分析河南、陕西分离的14株鸡杆菌(Gallibacterium)之间的进化关系,及gyrB基因序列比较在该菌进化分析中的作用。PCR扩增鸡杆菌分离株的gyrB、16SrRNA和rpoB3个看家基因,PCR产物纯化后直接测序。将鸡杆菌分离株、国外参考株的3个看家基因序列进行比较分析,用Phylip 3.67软件构建进化树。结果表明,14株鸡杆菌与鸭源鸡杆菌(Gallibacterium anatis)模式株间的相似性为96.3%～98.0%(gyrB)、97.7%～99.6%(16SrRNA)和97.7%～99.0%(rpoB);14株鸡杆菌与鸡杆菌复合群1(Gallibacterium genomosp.1)参考株间的相似性为88.8%～89.9%(gyrB)、96.2%～97.5%(16SrRNA)和92.6%～93.6%(rpoB)。基于3个看家基因序列的进化分析,均显示14株鸡杆菌和鸭源鸡杆菌模式株形成单独的一个群。14株鸡杆菌分离株均属于鸭源鸡杆菌种;在3个看家基因位点,鸡杆菌河南株与陕西株之间、鸡杆菌输卵管炎病鸡分离株与健康鸡分离株之间均无明显遗传上的差异;gyrB基因序列分析可用于鸡杆菌分离株的种类鉴定,且对14株鸡杆菌与复合群1参考株的区别能力优于另外2个看家基因。  相似文献   

16.
为考察恩诺沙星与乙酰甲喹的联合抗菌活性,采用肉汤稀释棋盘法测定了恩诺沙星与乙酰甲喹单用及联用对大肠杆菌、沙门氏菌临床菌株的体外抗菌活性。结果显示:恩诺沙星与乙酰甲喹单独使用对大肠杆菌的最小抑菌浓度为8μg/m L和32μg/m L,对沙门氏菌的最小抑菌浓度为16μg/m L和256μg/m L。两药联用对大肠杆菌和沙门氏菌的FIC指数为0.5和0.75,分别表现为协同作用和相加作用。试验表明,恩诺沙星与乙酰甲喹联用可以增强对大肠杆菌和沙门氏菌感染的治疗效果。  相似文献   

17.
Gallibacterium was recently established as a new genus including organisms previously reported as Pasteurella anatis, [Actinobacillus] salpingitidis and avian Pasteurella haemolytica-like organisms. The aim of the present study was to develop a PCR method allowing unambiguous identification of Gallibacterium. PCR primers positioned in the 16S rRNA (1133fgal) and 23S rRNA (114r) genes were defined and their specificity was subsequently tested on 122 strains. Twenty-five of the strains represented all of the presently available 15 phenotypic variants of Gallibacterium from different geographical locations, 22 other strains represented other poultry associated bacterial species or bacteria which could pose a differential diagnostic problem including members of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae, and finally 75 Gallibacterium field strains isolated from Mexican chicken egg-layers. Specific amplicons were generated in all 100 Gallibacterium strains tested, whereas none of the non-Gallibacterium strains tested positive. Correct identification was confirmed by hybridization with the Gallibacterium specific probe GAN850. Two internal amplification control strategies were successfully incorporated into the PCR assay, one based on amplification of the house-keeping gene rpoB (sharing target DNA) and another based on addition of trout DNA (foreign target DNA) and amplification with beta-actin specific primers. In conclusion, the described PCR assay enables specific identification of Gallibacterium and will thus stand as a strong alternative to the present diagnostic methods.  相似文献   

18.
The aim of this study was to identify lactic acid bacteria (LAB) using polymerase chain reaction (PCR) amplification of variable regions of the 16S rRNA gene. Thirteen LAB strains were isolated from the intestinal microbiota of healthy salmonids. A approximately 500-bp region of the highly conserved 16S rRNA gene was PCR-amplified and following this, a portion of the amplicon (272-bp) including the V1 and V2 variable regions was sequenced. The sequence containing both the V1 and V2 region provided strong evidence for the identification of LAB. The LAB strains were identified as Carnobacterium maltaromaticum, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, and Leuconostoc mesenteroides. The method described was found to be a very simple, rapid, specific, and low-cost tool for the identification of unknown strains of LAB.  相似文献   

19.
To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.  相似文献   

20.
Cat scratch disease (CSD) has been difficult to diagnose in animals because of the protracted clinical course of infection and the quiescent phases when the microbial culprit lies dormant. The causative agent in CSD appears to be multiple species and strains of Bartonella. Using polymerase chain reaction (PCR) techniques for amplification of highly variable regions of the 16S ribosomal RNA (rRNA) gene sequence, a very sensitive species- and strain-specific assay for CSD-causing Bartonella species was developed. PCR primers were designed to specifically amplify the 16S rRNA gene of Bartonella species but not of other microbial pathogens. This initial PCR was multiplexed with a universal primer set, based on conserved sequence regions in the 16S rRNA gene, that provides a 162-bp fragment in all species tested. Subsequently, 3 distinct nested PCR primer sets enabled the individual amplification and specific detection of Bartonella henselae type 1, B. henselae type II, and B. clarridgeae. Thus, this 2-step PCR procedure enabled the sensitive detection and identification of these species and the B. henselae genotype by exploiting minor sequences differences. Verification of these results were demonstrated with both sequencing and ligase chain reaction techniques. The diagnostic usefulness of this CSD test has been demonstrated by the analysis of specimens from control and infected cats. The diagnosis was confirmed and the specific B. henselae strain was correctly identified in peripheral blood specimens obtained from control and strain-specific CSD-infected cats. Such an accurate and sensitive diagnostic tool for the detection and identification of CSD causative agents should be a useful for the medical, veterinary, and scientific communities.  相似文献   

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