共查询到20条相似文献，搜索用时 31 毫秒
1.
Kang MS Kwon YK Jung BY Kim A Lee KM An BK Song EA Kwon JH Chung GS 《Veterinary microbiology》2011,147(1-2):181-185
Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgC and speC genes showing multiple mutations in the sequenced S. enterica subsp. enterica serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n=57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates. 相似文献
2.
Oliveira SD Santos LR Schuch DM Silva AB Salle CT Canal CW 《Veterinary microbiology》2002,87(1):25-35
A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level. 相似文献
3.
4.
根据鸡白痢沙门菌与鸡伤寒沙门菌的rfbS基因在第237和第598位碱基的不同，设计和合成了等位基因特异性PCR引物，建立了快速检测鸡白痢沙门菌的PCR方法，并应用该方法对鸡白痢沙门菌临床分离样品进行了PCR鉴定。结果显示，该PCR方法能够特异性地鉴定鸡白痢沙门菌，检测灵敏度达100PgDNA。对35个经常规方法鉴定的鸡白痢沙门菌分离株应用等位基因特异性PCR方法进行鉴定，鉴定出33株鸡白痢沙门菌，符合率为94．3％。表明，建立的等位基因特异性PCR方法能够准确而快速地鉴定鸡白痢沙门菌。 相似文献
5.
Okamura M Ohba Y Kikuchi S Suzuki A Tachizaki H Takehara K Ikedo M Kojima T Nakamura M 《Veterinary microbiology》2008,132(1-2):197-204
In the present study, the loop-mediated isothermal amplification (LAMP) assay was developed to amplify the fragments of the O9 Salmonella-specific insertion element and evaluated in the laboratory for its potential use in a field situation, such as poultry farms. Among the bacteria tested, a positive reaction was observed only for 128 strains of 6 serovars of the O9 group Salmonella, such as Enteritidis (SE) and Pullorum. The detection limit of the LAMP assay was 10(3)CFU/ml, which was more sensitive than that of the polymerase chain reaction (PCR) assay with the same target gene (10(6)CFU/ml). The final results were obtained within 30 min for the LAMP assay, while the PCR assay needed a total of 120 min. When the LAMP assay was applied to the enrichment broth mixed with cecal dropping samples either spiked with SE in vitro or excreted by SE-inoculated hens, the results were comparable to those of the conventional plating method including 2 separate enrichments. In conclusion, the LAMP assay developed in the present study is an effective method for the specific detection of the O9 group Salmonella serovars, including SE. 相似文献
6.
Amavisit P Browning GF Lightfoot D Church S Anderson GA Whithear KG Markham PF 《Veterinary microbiology》2001,79(1):63-74
A rapid polymerase chain reaction (PCR) assay was developed for detecting Salmonella in faeces of horses and assessed on samples from horses admitted to a veterinary hospital. Direct detection was achieved by amplification of part of ompC after extraction of DNA from faeces using a spin column method to reduce the amount of inhibitory substances in samples. An internal positive control was included to detect false negative results. While the sensitivity of the PCR assay was less than culture when assessed on faeces inoculated with Salmonella, its sensitivity on faecal samples obtained from horses was much greater than culture. Salmonella DNA was detected in 40% of faecal samples using the PCR assay while Salmonella were cultured from only 2% of the samples. The PCR assay has potential for use in either routine diagnosis or for detection of the carrier status in animals. 相似文献
7.
Contaminated poultry meat has been identified as one of the principal foodborne sources of Salmonella. The development of rapid detection assays for Salmonella would enable official agencies and food industries to identify contaminated foodstuffs in a more timely manner. In addition, these diagnostic tools could allow more 'real time' decisions to be made regarding end product acceptability. In this study, a survey was carried out to determine the prevalence of Salmonella in raw broiler carcasses. A total of 198 neck skin samples were obtained from within 40 flocks at a commercial broiler slaughtering facility. The presence of Salmonella was assessed by traditional culture methods and by a Salmonella-specific polymerase chain reaction (PCR) test. Salmonella was recovered from 32 (16%) of all samples using traditional culture methods. In contrast, the PCR assay proved to be more sensitive and detected Salmonella DNA in 38 (19%) of the samples tested. The pathogen was detected in 45 (23%) of the 198 samples when culture and PCR results were combined. The sensitivity of the PCR test was also greater than culture when detecting Salmonella from within flocks (53% of flocks by PCR, 30% of flocks by culture). The combination of both tests revealed that 55% of the flocks were contaminated with Salmonella. The PCR assay proved to be a highly specific and sensitive method for detecting Salmonella and the incorporation of a routine PCR test in conjunction with standard culture could be effective in providing a more accurate profile of the prevalence of this pathogen in broiler carcasses. 相似文献
8.
为鉴定新疆某些地区鸡白痢沙门氏菌主要流行株的流行现状和耐药情况,本研究采用平板凝集试验方法对有发病史的种鸡场进行现场检测,被检的750只鸡中,发现78例疑似鸡白痢沙门氏菌感染,运用沙门氏菌鉴别培养基、组织病理学观察和PCR方法进行确诊,并进行药敏试验。结果显示,疑似的78例鸡白痢沙门氏菌中有69例符合鸡白痢沙门氏菌的生化特性和病理学特点,PCR成功扩增出invA基因,分离率为9.2%,血清型诊断为D1型;其中分离菌对万古霉素和新霉素耐药性最高,耐药率分别为100.0%和81.2%,而对头孢曲松、克拉维酸和多黏菌素的敏感率达60.0%以上。本研究为养殖户和兽医工作者在净化鸡白痢沙门氏菌及临床用药方面提供参考。 相似文献
9.
LIU Zhi-ke YANG Ning-ning XU Jin-feng XU Ming-guo JING Ming-long WU Wen-xing CAO Xu-dong REN Yan SHI Feng CHEN Chuang-fu 《中国畜牧兽医》2017,44(9):2792-2800
In order to investigate the epidemic situation and drug resistance of major epidemic Salmonella Pullorum in some areas of Xinjiang, 750 chickens with disease history were detected using the methods of plate agglutination, and a total of 78 cases were diagnosed infection with Salmonella Pullorumin by using Salmonella identification medium, histopathological observation, PCR and drug sensitivity test.The results showed that 69 of 78 cases suspect Salmonella Pullorum were isolated, and prove the biochemical characteristics and pathologic characteristics of Salmonella Pullorum, the invA gene was amplified successfully by PCR,the isolation rate was 9.2% and the serotype was diagnosed as D1. The isolates were the most resistant to vancomycin and neomycin, and Salmonella Pullorum resistant rate was 100.0% and 81.2%, respectively. Drug sensitive test results showed that the sensitive rate of the bacteria were more than 60.0% to ceftriaxone,clavulanate and polymyxin. This study provided reference for farmers and veterinary workers, which indicated it could be used for the purification of Salmonella Pullorum and clinical medication. 相似文献
10.
Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples. 总被引：2，自引：0，他引：2
I S Schrank M A Mores J L Costa A P Frazzon R Soncini A Schrank M H Vainstein S C Silva 《Veterinary microbiology》2001,82(1):45-53
To attempt the rapid detection of Salmonella enterica, we have coupled a culture procedure with PCR amplification of the genus-specific invE/invA genes. The method was applied to different kinds of samples from the poultry industry and evaluated by using hydrolyzed feather meal, meat meal, litter and viscera, all experimentally inoculated with a known number of Salmonella followed by cultivation in selenite--cystine broth prior to the PCR reaction. The expected 457bp specific DNA fragment could be amplified from dilutions containing as few as 5.7CFU, indicating that the PCR technique can be successfully coupled with culture in an enrichment broth to distinguish Salmonella species from other enteric bacteria present in samples from the poultry industry. Tetrathionate broth proved to be a much better enrichment media compared to selenite-cystine when the presence of Salmonella was evaluated by PCR in 1-day-old chicks experimentally infected with known numbers of Salmonella. Samples included cecal tonsils and viscera, collected at 48h and 7 days postinfection. The PCR technique was more sensitive in detecting infected animals than the standard microbiological procedure, which detected only 47% of all PCR positive samples. 相似文献
11.
Hong Y García M Leiting V Bencina D Dufour-Zavala L Zavala G Kleven SH 《Avian diseases》2004,48(3):606-616
Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene-targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 x 10(2) color changing units/ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene-targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples. 相似文献
12.
本研究以单核细胞增生性李斯特菌Hly基因和InlA基因作为靶序列设计2对引物,建立了检测单增李斯特菌的二重PCR方法。结果表明,该方法可以同时扩增出单增李斯特菌的Hly基因和InlA基因,而对大肠杆菌、沙门氏菌等食品中的常见致病菌和英诺克李斯特菌均不能扩增出任何条带,表现出良好的特异性。二重PCR方法的最低检测限为104CFU/mL,并能够用于鉴定实验小鼠攻毒后内脏器官的单增李斯特菌分离培养物。该方法表现出良好的特异性、敏感性和通用性,适用于单增李斯特菌的快速鉴别检测。 相似文献
13.
14.
15.
J A Hasan I T Knight C R Tate E T Mallinson R G Miller R R Colwell S W Joseph 《Avian diseases》1991,35(2):397-402
A total of 48 environmental drag-swab samples from various poultry farms were tested for the presence of Salmonella spp. by culture, an enzyme-linked immunosorbent assay-based Salmonella antigen screening (SAS) assay, and two DNA probes (radiolabeled and colorimetric). The radiolabeled DNA probe was allowed to hybridize with culture-positive samples (n = 8) and was found to detect Salmonella spp. in all cases (100%). Both of the probes, subsequently hybridized with culture-negative samples (n = 8), were observed to yield good agreement (91%) with the culture findings. The remaining samples (n = 32) were tested by the SAS assay, and where there was no agreement between the culture and SAS, samples were further examined by the DNA probes. Results using both probes agreed with those obtained by culturing the samples but did not agree with the SAS assay result when the ratio of samples tested to samples positive (S/P) cutoff value used was 0.5. 相似文献
16.
A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1
A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 x 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission. 相似文献
17.
本研究采集某鸡场疑似感染鸡白痢的病死鸡组织,进行了沙门菌的分离、鉴定,以及对10种常见抗生素的药物敏感性试验。结果显示:分离株均为鸡白痢沙门菌,对阿米卡星(0%)、庆大霉素(0%)、头孢噻肟(3.70%)的耐药率较低,对链霉素(88.89%)、四环素(62.96%)、阿莫西林(51.85%)的耐药率较高;分离株至少可对1种药物产生耐药性,最多可对5种药物产生耐药性,70.37%的菌株可耐3种以上药物,其中耐3种药物的菌株数量最多(48.15%)。该研究结果不仅为临床合理用药提供指导,也为该地区鸡白痢的耐药性监测提供依据。 相似文献
18.
为比较不同方法检测鸡白痢沙门氏菌抗体消长的规律，以便为种鸡场净化提供科学指导，本研究以鸡白痢沙门氏菌活菌和灭活免疫原分别接种SPF鸡，采用平板凝集、微量凝集和ELISA试验定期检测血清中的特异性抗体，并以Kappa检验判定不同检测方法之间的一致性程度。结果显示，平板凝集试验在接种后检出抗体阳转的时间早于ELISA，但ELISA检出抗体阳性的持续时间更长，且更符合抗体消长规律；3种检测方法的结果之间仅具有微弱一致性（Kappa系数为0.002~0.295）。本研究结果表明，不同鸡白痢沙门氏菌抗体检测方法之间存在较大差异，但从总体来看，ELISA无假阳性干扰，检出抗体的持续时间较长，更符合抗体消长规律，在单一鸡白痢沙门氏菌感染的情况下，更有利于种鸡场对该病进行净化。 相似文献
19.
Carelli G Decaro N Lorusso A Elia G Lorusso E Mari V Ceci L Buonavoglia C 《Veterinary microbiology》2007,124(1-2):107-114
A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs. 相似文献
20.
为了探讨核苷酸结合寡聚化结构域1(nucleotide-binding oligomerization domain containing 1,NOD1)在抗沙门氏菌感染过程中的作用,本试验采用实时荧光定量PCR的方法检测了血液中NOD1基因在转录水平的表达量变化.试验分为3组,鸡白痢沙门氏菌组、肠炎沙门氏菌组和对照组,分别在感染后1、3、5、7 d检测NOD1 mRNA的表达水平.结果显示,感染后的1~7 d,鸡白痢沙门氏菌和肠炎沙门氏菌感染后的表达量变化趋势不同,其中,鸡白痢沙门氏菌感染后表达量呈先上升再下降然后再上升的波浪形变化,而肠炎沙门氏菌感染后的表达量呈逐渐上升趋势.与对照组相比,血液中NOD1 mRNA的水平,在鸡白痢沙门氏菌感染后的3和7 d的表达量显著高于对照组(P <0.05),在肠炎沙门氏菌感染后的5、7 d的表达量显著高于对照组(P <0.05).提示,鸡白痢沙门氏菌和肠炎沙门氏菌感染后均可促进鸡血液中NOD1基因的表达,NOD1基因可能参与了机体抗沙门氏菌感染过程. 相似文献