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1.
A Real-time quantitative PCR method was developed for the rapid detection of enterohemorrhagic Escherichia coli （EHEC） O157∶H7 based on the primers and TaqMan probes which were designed for the conservative domain of rfbE gene of EHEC O157∶H7.The results showed that the sensitivity of Real-time PCR was 7.3 CFU/mL.20 from 310 samples of meat,egg,milk and its products,animal diarrhea materials and artificial contamination samples were positive detected by Real-time PCR assay,which was in accordance with the testing result according to AOAC standard.The results indicated that Real-time PCR assay was a sensitive,rapid and simple tool. 相似文献
2.
Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea 总被引：3，自引：0，他引：3
Lim SK Joo YS Moon JS Lee AR Nam HM Wee SH Koh HB 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2004,66(5):581-584
One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea. 相似文献
3.
多重PCR快速检测奶牛乳房炎3种主要病原体 总被引：10，自引：0，他引：10
奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。 相似文献
4.
应用环介导等温扩增技术快速检测单核细胞增生李斯特菌的研究 总被引：1，自引：0，他引：1
目的建立环介导等温扩增技术快速检测单核细胞增生李斯特菌。方法根据单核细胞增生李斯特菌(LM)hlyA基因序列中的保守区域,采用在线引物设计软件Primer Explorer4.0进行设计,获得一套特异性的环介导等温扩增(LAMP)引物,对单核细胞增生李斯特菌hlyA基因进行LAMP扩增,并与常规PCR方法进行比较。结果建立的LAMP方法能成功扩增出梯形条带,LAMP检测单核细胞增生李斯特菌纯培养物和人工染菌的灵敏度为5.44×102cfu/mL,而对照PCR检测的灵敏度为5.44×104cfu/mL。对10株细菌进行LAMP扩增,仅单核细胞增生李斯特菌得到阳性结果。从DNA提取到报告结果,耗时仅1h。结论 LAMP检测单核细胞增生李斯特菌灵敏度高,特异性强,耗时短,方法简便,有望发展成为快速检测食品中单核细胞增生李斯特菌的有效手段。 相似文献
5.
Kitai S Shimizu A Kawano J Sato E Nakano C Kitagawa H Fujio K Matsumura K Yasuda R Inamoto T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(3):269-274
A total of 444 samples of raw chicken meat (thighs, breasts, wings, livers, gizzards, hearts and ovaries) that retailed at 145 different supermarkets in 47 prefectures in Japan were examined for contamination with Staphylococcus aureus in association with its enterotoxigenicity. S. aureus was isolated from 292 (65.8%) of the samples, and from 131 of the 145 supermarkets. There was no significant difference in the detection rate of S. aureus according to the type of meat examined. About 80% of 714 isolates belonged to the poultry (57.1%) and human biotypes (22.1%). Seventy-eight (21.7%) of 360 isolates were enterotoxigenic and isolated from 78 samples in 53 supermarkets in 31 prefectures. Staphylococcal enterotoxins (SEs) produced were SEB (50 isolates), SEA (14), SEC (8), SED (2), SEA+SEB (2), and SEA+SEC (2). Most of the enterotoxigenic isolates belonged to the human and poultry biotypes, coagulase type VII, VIII or IV, and were lysed by phages of group III. Identical SE types, biotypes, coagulase types and pulsed-field gel electrophoresis (PFGE) patterns were shown in isolates from different types of meat at the same supermarket and from samples taken from different supermarkets in the same prefectures or in isolates from samples obtained from several different prefectures. Among the 50 SEB-producing isolates, 27 yielded three similar PFGE patterns that differed by only a few fragments, suggesting that they were closely related genetically. The three patterns were found in isolates of samples that retailed at 17 supermarkets in 11 prefectures, indicating that they may be disseminated among raw chicken meat in Japan. 相似文献
6.
检测K88~+肠毒素性大肠杆菌PCR方法的建立 总被引：5，自引：0，他引：5
以K8 8菌毛结构基因保守序列为靶序列 ,设计合成了一对可扩增长 2 0 1bp的目的片段的引物 ,成功地建立了检测肠毒素性大肠杆菌(ETEC)K88菌毛基因的PCR方法。进行了PCR方法的特异性试验和敏感性试验。对K99+ ,F41 + ,987p+ 参考菌株和鼠伤寒沙门氏杆菌 ,链球菌 ,金黄色葡萄球菌和猪肺疫巴氏杆菌的检测结果均为阴性 ;该检测方法的敏感度可达 1 0个细菌。用此方法对 1 0株腹泻仔猪粪便分离物进行检测 ,结果有 2株阳性 ;与血清学检测的结果一致。结果表明此方法特异性和敏感性都很高 ,可用于临床K88+ 肠毒素性大肠杆菌病的快速诊断和流行病学调查 相似文献
7.
鸡传染性支气管炎病毒的快速检测 总被引：4，自引：0，他引：4
本试验建立了一种快速检测鸡传染性支气管炎病毒的通用性PCR方法。通过对纯化后IBV核蛋白基因进行的直接PCR及套式PCR扩增表明，两次PCR产物的大小为0.7kb和0.5kb，与实际设计相符。而对照的NDV主IBDV的PCR扩增产物均为阴性。用IBV HB（华北）株感染SPF鸡后，应用我们所建立的PCR方法去检测不同时期所采集的肾脏、气管、盲肠扁桃体、肺脏和肝脏中的IBV，在SPF鸡感染IBV后的21天仍然能够检测到IBV的基因组，Southern blot杂交试验证明了我们获得的PCR产物为IBV的核蛋白基因。对30份自然感染病料的检测表明，本实验所建立的PCR方法检测阳性数为15份，阳性率为50％（15／30）；鸡胚接种检测的阳性数为17份，阳性率为56．7％（17／30）；IFA检测的阳性数11 ，阳性率为36．7％（11／30）。PVR检测法与鸡胚接种方法的阳性符合率为93．3％（14／15），统计学分析表明，P＞0．05，差异不显著，而PCR检测法与IFA的阳性符合率为60％（9／15）。结果证明本实验所建立的PCR检测方法具有敏感、特异、快速等特点，适用于不同来源及不同血清型IBV的检测。 相似文献
8.
检测伪狂犬病毒gB基因荧光定量PCR方法的建立 总被引：1，自引：0，他引：1
利用DNAMAN软件对GenBank登录的伪狂犬病病毒（PRV）各毒株gB基因序列进行比对分析,选择其保守区域设计合成特异性的引物和TaqMan探针,同时利用普通PCR技术扩增获得全长的伪狂犬病毒gB基因,并克隆到pMD20-T载体上作为阳性标准品。通过对荧光定量PCR反应条件的优化,建立了一种快速检测伪狂犬病病毒的荧光定量PCR技术。该检测技术具有较高的灵敏性、特异性和可靠性。对制备的pMD-gB阳性标准品的检测结果表明,所建立的伪狂犬病毒TaqMan荧光定量PCR最低检测极限可达1.50×102拷贝/反应;同时相比于普通PCR方法其灵敏度高100～1 000倍以上,并且重复性好。在对60份临床样品的检测中,荧光定量PCR不仅检出了普通PCR检测为阳性的样品,且检出了2份普通PCR未检出的样品,进一步证实了该方法快速、灵敏性好,可用于PRV感染的早期快速定量检测和肉类食品进出口检疫。 相似文献
9.
为了建立快速简便的fneB-LAMP检测法用于马腺疫病原马链球菌马亚种(S.equi)的检测,本试验采用恒温水浴进行LAMP扩增,建立可视化fneB-LAMP检测方法.结果显示:对大肠埃希菌、无乳链球菌、金黄色葡萄球、蜡状芽孢杆菌和克雷伯杆菌5种菌与标准S.equi菌株检测,只能够检测出标准阳性菌株;对新疆昭苏县3个马... 相似文献
10.
为了建立猪链球菌(Streptococcus suis,SS)种与9型猪链球菌(SS9)的快速诊断方法,本研究根据GenBank已登录的SS种特异性基因gdh和SS9型特异性基因CPS9H设计引物,以标准SS9株基因组DNA为模板,建立了SS种和SS9的二重PCR检测方法,并进行了特异性、敏感性和重复性试验;利用所建立的方法对检测疑似猪链球菌感染猪临床样品,并与常规细菌分离鉴定方法进行了比对。结果表明成功建立SS种和SS9型猪链球菌二重PCR检测方法,该方法的检测灵敏度可达100个CFU,特异性和重复性好;利用该方法对34份临床分离自疑似猪链球菌感染样品的细菌培养物进行了应用检测试验,其中有11份样品为gdh阳性,11份gdh阳性样品中有3份样品同时为SS9阳性。本研究成功建立了SS种与SS9型猪链球菌二重PCR检测方法,可用于猪链球菌种和SS9型猪链球菌的快速诊断。 相似文献
11.
Rall VL Vieira FP Rall R Vieitis RL Fernandes A Candeias JM Cardoso KF Araújo JP 《Veterinary microbiology》2008,132(3-4):408-413
Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety. 相似文献
12.
为了解引起山羊皮下脓肿的主要病原,本研究从四川乐至县、金堂县和蓬溪县3个山羊养殖场无菌采集患皮下脓肿病羊脓液样本19份,通过细菌分离培养、生化试验及特异性PCR方法对其病原进行检测和鉴定,并通过药敏试验测定分离病原的药物敏感性。结果显示,PCR扩增法共检测出病原菌19株,其中伪结核棒状杆菌、金黄色葡萄球菌及化脓隐秘杆菌分离率分别为57.9%(11/19)、26.3%(5/19)及15.8%(3/19);采用细菌分离培养法分离到3种形态不同的病原菌共计17株,其中革兰氏阳性短杆菌经鉴定为伪结核棒状杆菌,分离率57.9%(11/19),革兰氏阳性球菌经鉴定为金黄色葡萄球菌,分离率为21.1%(4/19),革兰氏阳性杆菌经鉴定为化脓隐秘杆菌,分离率为10.5%(2/19),PCR鉴定较细菌分离培养法灵敏度更高。3种病原菌对丁胺卡那、氟苯尼考等多种药物敏感。结果表明,伪结核棒状杆菌是引起该地区山羊皮下脓肿的主要病原,金黄色葡萄球菌和化脓隐秘杆菌同样可引起山羊皮下脓肿,特异性PCR方法较细菌分离培养更为高效、准确。本研究结果可为后续进一步分析其生物学特性,以及四川地区山羊皮下脓肿的综合性防制提供依据。 相似文献
13.
Enterotoxigenic Staphylococcus aureus in raw milk poses a potential health hazard to consumers, and the identification of such strains should be used as part of a risk analysis of milk and milk products. The primary purpose of this study was to investigate the occurrence of enterotoxigenic S. aureus strains in raw milk supplied for dairy processing in the Czech Republic. A further aim was to compare the production of staphylococcal enterotoxins (SEs) with the presence of the corresponding genes. This was undertaken using multiplex polymerase chain reaction (PCR) and reversed passive latex agglutination (RPLA). Out of 440 bulk tank milk samples from 298 dairy herds, 70 proved positive for S. aureus (15.9%). Staphylococcal enterotoxin genes (ses) were detected in 39 (55.7%) isolates. The genes most commonly detected were sei (38.6%), seg (31.4%) and sea (27.1%). Genes seb, seh, sed, sej and sec were observed in 10%, 4.3%, 2.9%, 2.9% and 1.4% of strains respectively. Genes see and sel did not occur. The most frequently detected genotypes were seg, sei at 11.4%; sea at 10.0%; and sea, seg, sei at 8.6%. Toxin production was observed in nine (12.9%) S. aureus isolates. Seven strains were detected as SEB- (10%) and two as SED- (2.9%) producing. A relatively high number (32%) of discrepancies between the results with multiplex PCR and RPLA assays was obtained, particularly on account of SEA. Nineteen strains were sea positive by PCR but SEA negative by RPLA, and one strain was sec positive and SEC negative. The results of both methods were identical concerning SEB and SED. It was concluded that detection of ses by PCR was a useful additional tool to support identification of enterotoxigenic strains. 相似文献
14.
为了解哈尔滨市单核细胞增生性李斯特茵(Lm)的污染状况及耐药状况.在哈尔滨市市场随机采集158份鲜肉样品,采用显色培养基分离,API试剂条和PCR鉴定等方法对样品中的Lm进行分离鉴定,并通过Kirby-Barer法测定分离菌株对24种抗生素的耐药性.结果从鲜肉中共分离到Lm 23株,检出率为14.56%,其中鲜猪肉检出率最高,达20.00%(14/70);23株分离菌株中耐药菌株为22株,耐药率高达95.65%.这表明哈尔滨市鲜肉中存在一定程度的Lm污染,并且分离菌株存在较严重的耐药现象.应加强控制动物饲料亚治疗抗生素的使用并严格遵守休药期,防止耐药菌株产生进而控制食源性疾病的发生. 相似文献
15.
Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds 总被引：2，自引：0，他引：2
To determine the distribution of genes that encode enterotoxins A, B and C, 36 strains of Staphylococcus aureus isolated from goat mastitis and 64 isolated from bovine mastitis were analyzed by Multiplex PCR. Of the total strains studied, 37 (37%) were detected to have some of the SEs genes. From the bovine mastitis strains, 4 (6.3%) co-amplified the sea and seb genes and 2 (3.1%) were positive for the sec gene. From the goat mastitis strains, 31 (86%) tested positive to the Multiplex, and the sec gene was detected in all of them. The production of SE was detected in all strains harboring the corresponding gene. The results demonstrated that S. aureus isolated from goat mastitis had a higher enterotoxigenic potential than those isolated from bovine mastitis. Additionally, the presence of the sec gene in the majority of goat mastitis strains suggests a possible involvement of SEC in goat mastitis pathogenesis. 相似文献
16.
17.
依据GenBank中登录的常见细胞污染支原体16S rRNA基因序列,选择高度保守的区域设计引物,建立了一种快速灵敏的支原体PCR检测方法.该方法可以特异性检测出6种支原体,并能敏感的检测到10个拷贝数的目的基因.采用建立的PCR方法和传统的培养法对23个不同样品(细胞、血清、疫苗成品、半成品)进行检测,符合度100％.该方法的建立可大大缩短疫苗生产过程中支原体检验所需时间. 相似文献
18.
Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully. 相似文献
19.
The aim of this study was to establish a simultaneous triple PCR detection method for Staphylococcus aureus (S.aureus),Pseudomonas aeruginosa (P.aeruginosa) and Klebsiella pneumoniae (K.pneumoniae).Three pairs of specific primers had been designed according to nuc gene of S.aureus,toxR gene of P.aeruginosa and PhoE gene of K.pneumoniae.The triple PCR reaction conditions were optimized on the basis of single PCR methods.At the same time,specificity,sensitivity and repeatability tests of the triple PCR method were studied,and the results of bacteria isolation and culture and the triple PCR method were compared.The results showed that the amplification product sizes were 484,278 and 368 bp,respectively.The optimal annealing temperature was 56 to 59 ℃,the concentrations of primers were all 0.2 μmol/L,dNTP concentration was 200 μmol/L,Mg2+ concentration was 2.5 mmol/L.The specificity test showed that there was no cross reaction between these three bacteria templates and other eight kinds of common bacteria templates,such as Bordetella bronchiseptica.The minimum of simultaneous detection of three bacteria genomic DNA was 10-5 ng/μL.The results of three repeatability tests of triple PCR were the same which indicated the repeatability was good.30 samples from mice were detected by bacteria isolation and culture and the triple PCR method,the detection rate of triple PCR was slightly higher than the bacteria isolation and culture.The positive samples detected by bacteria isolation and culture were also positive detected by triple PCR.The results showed that a specific,sensitive and efficient triple PCR system had been established and could provide the technical support for bacteria detection and epidemiological investigation of laboratory animals. 相似文献
20.
本研究旨在建立一种能同时检测金黄色葡萄球菌(Staphylococcus aureus,S.aureus)、绿脓杆菌(Pseudomonas aeruginosa,P.aeruginosa)、肺炎克雷伯杆菌(Klebsiella pneumoniae,K.pneumoniae)的三重PCR检测方法.根据金黄色葡萄球菌nuc基因、绿脓杆菌toxR基因、肺炎克雷伯杆菌PhoE基因设计并合成引物,在单一PCR条件基础上优化建立三重PCR反应条件,并进行特异性、敏感性和重复性分析及与细菌分离培养的比对试验.结果显示,3对引物均能特异性扩增出目的条带,大小分别为484、278和368 bp.最佳退火温度在56~59 ℃之间,引物浓度均为0.2 μmol/L,dNTP浓度为200 μmol/L,Mg2+浓度为2.5 mmol/L.3种细菌间无交叉反应.对支气管鲍特杆菌等其他8种实验动物常见致病菌均无交叉反应.最低能同时检测到10-5 ng/μL的细菌基因组DNA.3次重复结果一致,表明建立的三重PCR方法重复性好.同时采用细菌分离培养法和三重PCR方法对30份实验小鼠样本进行检测,对比结果显示三重PCR方法检出率略高于细菌分离培养法,细菌分离培养法呈阳性的样品,三重PCR方法均能检出.结果表明,本试验建立的三重PCR检测方法具有特异、敏感、高效等优点,为实验动物细菌快速检测和流行病学调查提供了技术支持. 相似文献