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1.
Since the first isolation of Helicobacter pylori from humans in 1983, 18 Helicobacter species have been identified during the last decade in domestic and laboratory animals. Several Helicobacter species have been isolated from the gastrointestinal tracts of various mammalian species and birds. Helicobacter hepaticus, H. muridarum, H. bilis, H. rodentium and Flexispira rappini have been isolated from mice. Among these species, only H. hepaticos has been clearly recognized as a pathogen. Indeed, it displays the pathogenic potential to elicit hepatitis in several strains of mice; moreover in A/JCr mice, it is strongly associated with hepatic cancer. Among the five murine helicobacter species, apart from H. hepaticus, F. rappini has not been found associated with lesions, H. muridarum has been observed in gastric glands of mice with chronic gastritis, and H. bilis has been reported in the liver of mice with chronic hepatitis. When associated with H. rodentium, H. bilis is able to induce diarrhea in SCID mice. In no case has pathogenicity of a single species been clearly proven. In rats, H. trogontum and H. muridarum have been isolated from the intestine, without any information concerning their respective pathogenicity. H. cinaedi and H. cholecystus have been identified from the intestine and the gallbladders of hamsters, respectively. The diagnosis of Helicobacter species by polymerase chain reaction (PCR) is a rapid, specific and sensitive technique. One of the most promising diagnostic techniques of these infections seems to be the PCR detection of Helicobacter sp. from feces based on the 16S rRNA sequences, then a restriction enzyme analysis to identify the actual species. Several drug regimens have also been evaluated to eradicate H. hepaticus from mice. Helicobacter infections, particularly H. hepaticus and H. bilis, seem to be widespread in laboratory mouse colonies and have also been detected from commercial breeders. 相似文献
2.
This study examined the prevalence of Helicobacter infection in the pyloric mucosa of pigs and its histopathological and molecular characteristics. Forty porcine pyloric samples were examined for Helicobacter infection by silver staining and PCR assay. The PCR product (376 bp) was digested with NdeII to differentiate between Helicobacter heilmannii and Helicobacter pylori. Another PCR assay run to produce an 1157 bp fragment was performed using a primer set designed from the 16S rRNA gene of Candidatus H. suis, and its product was cloned and sequenced. Infection rates were 62.5% (25/40) and 95.0% (38/40) as determined by silver staining and the PCR assay, respectively. On histopathological examination, lymphoid follicle aggregation in the pyloric mucosa and granulocytic migration into the lumen of pyloric glands were observed in 24 (60.0%) and 33 (82.5%) gastric samples, respectively. All PCR products, except that of H. pylori, were cut into two fragments of 147 and 229 bp by enzymatic digestion with NdeII. Sequencing of the 16S rRNA gene showed that the bacterium had 99.57% (1152 bp/1157 bp) homology to the 16S rRNA gene of Candidatus H. suis. 相似文献
3.
根据肝螺杆菌fla B基因保守区域设计一组特异性引物,经过条件优化、特异性和敏感性分析,成功建立了肝螺杆菌LAMP快速检测方法。结果显示,建立的LAMP扩增方法仅能检测出肝螺杆菌,其他常见细菌未见特异性扩增。检出肝螺杆菌最低模板量为86.9fg/L,是普通PCR所需模板量的1/10。本研究建立的LAMP快速检测方法具备操作简单、特异性好、灵敏度高的优点,结果易于观察,对仪器设备要求低,适宜推广应用。 相似文献
4.
Neubauer C Hess M 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2006,53(8):376-381
The objective of this study was to develop a multiplex polymerase chain reaction (PCR) to detect and differentiate food-borne pathogens of the three genera Campylobacter, Arcobacter and Helicobacter in a single step procedure. One common reverse primer and three genus-specific forward primers were designed by hybridizing to the 16S rRNA of selected reference strains. Besides the species with significance as food-borne pathogens isolated from poultry meat--Campylobacter jejuni, Campylobacter coli, Arcobacter butzleri and Helicobacter pullorum--several other members of these genera were tested to determine the specificity of the designed multiplex PCR. In total, 20 ATCC and NCTC reference strains of Campyobacter, Arcobacter and Helicobacter were used to evaluate the PCR. Specific amplificates were obtained from all thermophilic species of Campylobacter as well as from species of Arcobacter and Helicobacter. No amplification product was obtained from the non-thermophilic Campylobacter, C. hyointestinalis and C. fetus. Furthermore, a total of 43 field strains of the three genera isolated from poultry, pigs, cattle and humans were investigated using this PCR. To confirm the classification of 10 H. pullorum strains the 16S rRNAs were sequenced. The developed PCR is a helpful diagnostic tool to detect and differentiate Campylobacter, Arcobacter and Helicobacter isolated from poultry and poultry products. 相似文献
5.
为对鼠源细胞进行鉴别检测,以鼠线粒体16S rRNA基因序列为靶位点设计特异性引物及探针,建立实时荧光定量PCR检测方法,并评价该方法的特异性及敏感性。结果显示,所建立的检测方法特异性好,针对鼠源细胞基因组荧光定量PCR扩增曲线良好,其他物种来源细胞基因组及生物制品原辅材料未出现特异性扩增曲线;敏感性高,基因拷贝数检出限度为45.3拷贝。本试验建立的荧光定量PCR检测方法能够有效地对鼠源细胞进行快速检测,为细胞质量控制提供了有效方法。 相似文献
6.
支原体PCR检测方法的建立及初步应用 总被引：1，自引：1，他引：0
经过对Genebank鸡滑液支原体、猪肺炎支原体、口腔支原体和猪鼻支原体16S rRNA序列比对,设计了一条通用引物,建立支原体PCR检测方法.该方法敏感、特异、经济、快速,在动物疫苗生产中检测细胞、半成品及成品支原体污染具有较大的应用价值. 相似文献
7.
Van den Bulck K Baele M Hermans K Ducatelle R Haesebrouck F Decostere A 《Veterinary research communications》2005,29(4):271-279
Gastric Helicobacter spp. have been described in a wide range of animal species, including dogs, cats, primates, swine, cattle and rodents. However, in lagomorphs--more specifically rabbits--gastric Helicobacter infections have never been reported. Biopsy specimens were collected from different stomach regions of 23 rabbits, including 10 pet rabbits, 10 industrial animals and 3 research animals. These were subjected to a PCR assay for the detection of Helicobacter DNA. Identification up to the species level was based on 16S rRNA sequence analysis and a recently developed multiplex PCR. Seven rabbits (four pet, one research animal and two industrial animals) tested positive in the Helicobacter genus-specific PCR in the stomach, with the corpus being predominantly positive. H. felis and H. salomonis, hitherto presumed to be naturally hosted by cats and dogs, were detected in three animals and one animal, respectively. One of these animals had been completely devoid of any form of contact with cats or dogs. A H. pullorum/H. rappini-like organism (96% 16S rDNA sequence similarity) was found in an industrially held rabbit. The helicobacters of the two remaining rabbits could not be identified up to the species level. To conclude, this is the first report on the occurrence of Helicobacter spp. in the stomach of rabbits. In view of the fact that H. felis and H. salomonis are put forward as having zoonotic potential, further research is necessary to investigate the implications of these findings not only for the rabbit but also for human health. 相似文献
8.
Criado-Fornelio A Rey-Valeiron C Buling A Barba-Carretero JC Jefferies R Irwin P 《Veterinary parasitology》2007,144(3-4):261-269
The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil. 相似文献
9.
根据猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)的16S rRNA基因设计3条引物, 建立Mhp和Mhr的双重PCR检测方法,并对该方法进行了特异性和敏感性试验,并使用建立的方法检测了临床样品和疫苗样品。结果显示该方法具有良好的特异性,最低可检测到0.66ng 的Mhp基因组DNA和0.58 ng Mhr基因组DNA,临床样品和疫苗样品检测结果与普通PCR检测结果一致。该双重PCR方法,可用于Mhp与Mhr的鉴别、诊断以及疫苗纯粹性检查,快速而准确。 相似文献
10.
根据GenBank上发表的猪附红细胞体16S rRNA基因序列(登录号U88565)设计合成2对引物,建立了猪附红细胞体单管巢式PCR诊断方法,经酶切分析、单管巢式PCR进一步鉴定后进行序列测定,并与血涂片染色镜检、常规PCR进行了比较。结果:扩增的猪附红细胞体基因序列与GenBank中发表的猪附红细胞体基因序列(U88565)同源性为96%;特异性试验表明,设计的引物不能扩增弓形虫、链球菌、大肠杆菌、葡萄球菌及羊附红细胞体等病原体;敏感性试验表明,单管巢式PCR诊断方法最低能够检测出0.116 fg的标准模板DNA。通过对75份血液样本的检测表明,建立的单管巢式PCR方法明显优于血涂片染色镜检法及常规PCR方法,具有较高的敏感性和实用性。本试验建立的单管巢式PCR诊断方法具有特异、敏感、实用等优点,为猪附红细胞体的检测提供了一种新型、可靠的诊断技术。 相似文献
11.
Buckley TC Millar BC Egan CL Gibson P Cosgrove H Stanbridge S Matsuda M Moore JE 《Irish veterinary journal》2005,58(3):146-149
: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes. 相似文献
12.
《Veterinary microbiology》1997,58(1):23-30
The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods. 相似文献
13.
Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection. 总被引：17，自引：0，他引：17
The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. The gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectively amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gene from DNA of E. suis extracted from the blood of an experimentally infected pig during a parasitemic episode. No polymerase chain reaction (PCR) products were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were observed with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using PCR reagents without target DNA. The detection limits for E. suis by competitive quantitative PCR were estimated to range from 57 and 800 organisms/assay. This is the first report of the utility of PCR-facilitated diagnosis and quantitation of E. suis based on the 16S rRNA gene. The PCR method developed will be useful in monitoring the progression and significance of E. suis in the disease process in the pig. 相似文献
14.
细菌通用引物在奶牛乳房炎病原菌检测中的作用研究 总被引：3，自引：0，他引：3
为了建立PCR直接检测奶牛乳房炎致病菌的方法,探索通用引物在奶牛乳房炎致病菌检测中的应用价值。利用16S rRNA基因的高度保守性,设计并合成细菌的通用引物,采用合成的引物扩增标准菌株及患有奶牛乳房炎的奶样。结果表明,通用引物扩增7 种标准菌株,370 bp处均可得到清晰的电泳条带;通用引物PCR 可检出250 ng/L的金黄色葡萄球菌标准菌株DNA;对患有奶牛乳房炎的奶样进行扩增,370 bp处也得到了清晰的电泳条带。建立在16S rRNA基础上的通用引物在奶牛乳房炎的检测中,初步显示具有特异、快速等优点,为进一步判断细菌的种类奠定了基础。 相似文献
15.
A wild-type isolate with similar morphological and phenotypic properties to Taylorella equigenitalis, the causative bacterial agent of contagious equine metritis (CEM), was referred for molecular identification by PCR amplification of the 16S rRNA gene. A species-specific PCR failed to yield a product compatible with that of T. equigenitalis. The direct sequencing of the universal 16S rRNA PCR amplicon suggested the presence of a Bacteroides sp., probably Bacteroides ureolyticus, with no consequent effects on the movement and transportation of the animal. Adoption of such a molecular means of identification through sequencing may aid in the identification of the atypical forms of Taylorella equigenitalis, as recently described, as well as differentiating this species from Taylorella asinigenitalis. 相似文献
16.
17.
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
18.
本研究旨在建立小亚璃眼蜱、亚洲璃眼蜱和残缘璃眼蜱的分子生物学鉴定方法,并探讨它们的系统发生关系。在新疆、内蒙古从动物体表采集寄生蜱,形态学鉴定后,PCR扩增测序获得3种璃眼蜱的16S rRNA及线粒体色素氧化酶亚基Ⅰ基因(cytochrome oxidaseⅠ,COⅠ)序列后进行同源性分析。用Mega 5.0和Mrbayes 3.2软件分别构建系统进化树,亚洲璃眼蜱、小亚璃眼蜱样本16S rRNA序列与GenBank中已知相应蜱虫16S rRNA序列聚类,而残缘璃眼蜱COⅠ序列与GenBank中已知残缘璃眼蜱COⅠ序列聚类,与形态学鉴定结果一致。在传统形态学分类的基础上,结合分子生物学鉴定方法能简易、准确鉴定小亚璃眼蜱、亚洲璃眼蜱和残缘璃眼蜱。 相似文献
19.
Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates 总被引：2，自引：0，他引：2
Takahashi-Omoe H Omoe K Matsushita S Kobayashi H Yamamoto K 《Comparative immunology, microbiology and infectious diseases》2004,27(2):117-128
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.
J M Miller A L Jenny J L Ellingson 《Journal of veterinary diagnostic investigation》1999,11(5):436-440
A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections. 相似文献