用抗体致敏SPA菌体浓缩新城疫病毒方法的建立

将金黄色葡萄球菌与灭活的猪抗新城疫病毒(NDV)抗血清混合，37C感作致敏30min，将致敏的金黄色葡萄球菌加入NDV污染材料洗脱液中，37C水浴30min，离心洗涤并悬浮菌体于PBS中，取适量用于电镜观察或RTPCR。结果显示，电镜下观察可见NDV吸附到金黄色葡萄球菌表面；经RT—PCR对吸附NDV的菌体进行扩增后可见到特异性扩增带。通过条件优化，建立了NDV浓缩方法。用HA效价为2^9的NDV F48E9毒株尿囊液1：800、1：1600和1：3200稀释液分别喷洒树叶及谷物，采用上述方法浓缩病毒，并经RT—PCR检测。结果表明，致敏的金黄色葡萄球菌能够浓缩病毒，提高RT—PCR检测的敏感性。     

特异抗体致敏的SPA菌体浓缩新城疫病毒方法在临床诊断中的应用

为提高新城疫临床诊断的准确率,建立了新城疫病毒浓缩方法,将粪便及脑、肺等器官组织病料中的新城疫病毒经特异性抗体致敏的金黄色葡萄球菌(SPA菌)吸附浓缩后,用RT-PCR进行检测。结果表明,所建立的新城疫病毒浓缩方法应用于临床诊断后,可显著提高检测敏感性,从而提高临床诊断的准确率。     

特异抗体致敏的SPA茵体浓缩新城疫病毒方法在临床诊断中的应用

为提高新城疫临床诊断的准确率,建立了新城疫病毒浓缩方法,将粪便及脑、肺等器官组织病料中的新城疫病毒经特异性抗体致敏的金黄色葡萄球菌(SPA菌)吸附浓缩后,用RT-PCR进行检测.结果表明,所建立的新城疫病毒浓缩方法应用于临床诊断后,可显著提高检测敏感性,从而提高临床诊断的准确率.     

食品中金黄色葡萄球菌PCR检测方法的建立

采用PCR技术特异性扩增金黄色葡萄球菌的耐热核酸酶编码基因(nuc基因),检测模拟样品中金黄色葡萄球菌。结果表明所建立的模拟食品中金黄色葡萄球菌的PCR检测方法,特异性良好,敏感性可达61.76pg/μl。     

荧光定量PCR检测牛乳中金黄色葡萄球菌肠毒素A基因方法的建立

为建立检测牛乳中金黄色葡萄球菌(S.aureus)肠毒素A基因(SEA)定性定量的检测方法,本研究针对S.aureus SEA基因片段设计1对引物,将构建的重组质粒作为阳性对照,建立了S.aureus SEA DNA的SYBR Green I real-time PCR检测方法.结果显示,特异性产物Tm值为78.2℃～78.5℃,最低可检测到49.5 fg/μL(16.5拷贝)的阳性质粒.标准曲线的相关系数为0.99.与其他常见的产SEB的S.aureus、产SEC的S.aureus、无乳链球菌、大肠杆菌、嗜热链球菌、伤寒沙门氏菌、大肠杆菌DH5 α及JM109均无交叉反应.该检测方法具有较好的特异性和敏感性,为牛乳中S.aureus的快速检测提供了新的技术手段.     

PCR技术在鼠金黄色葡萄球菌检测中的初步研究

根据已公布的金黄色葡萄球菌耐热核酸酶nuc基因的序列 ,设计并合成一对特异性的引物 ,利用PCR技术扩增nuc基因片段。对金黄色葡萄球菌和其他非金黄色葡萄球菌菌株抽提的DNA进行扩增。结果金黄色葡萄球菌PCR产物出现 6 6 8bp的特异性DNA扩增片段 ,而其他非金黄色葡萄球菌未出现扩增片段 ,证实了合成的引物对金黄色葡萄球菌具有特异性。将抽提的金黄色葡萄球菌DNA进行系列稀释 ,测定此PCR体系的敏感性。结果显示 ,该PCR体系能检出 3pg金黄色葡萄球菌DNA ,且从抽提DNA到PCR扩增及电泳结束仅需 4h。因此 ,研究所建立的扩增耐热核酸酶nuc基因检测鼠金黄色葡萄球菌的PCR方法 ,具有快速、可靠、敏感和特异的特点 ,可用于临床样品和金黄色葡萄球菌感染时的检测 ,适合应用于实验大小鼠的监测     

一种简便的热启动PCR方法扩增金葡菌nuc基因

本文采用低熔点琼脂糖包被Taq酶热启动聚合酶链式反应(PCR)扩增出金黄色葡萄球菌耐热核酸酶(nuc)基因,该法与普通PCR扩增效果无异且具有避免非特应性扩增等特点.同时研究了不同退火温度对扩增反应结果的影响,确定了51.7C为最佳退火温度.为进一步研究热启动PCR方法提供了依据.     

一种简便的热启动PCR方法扩增金荀菌nuc基因

本文采用低熔点琼脂糖包被Taq酶热启动聚合酶链式反应（PCR）扩增出金黄色葡萄球菌耐热核酸酶（nuc）基因,该法与普通PCR扩增效果无异且具有避免非特应性扩增等特点。同时研究了不同退火温度对扩增反应结果的影响,确定了51．7℃为最佳退火温度。为进一步研究热启动PCR方法提供了依据。     

奶牛乳房炎金黄色葡萄球菌和大肠杆菌二重PCR检测方法的建立

金黄色葡萄球菌和大肠杆菌是引起奶牛乳房炎的主要病原菌。选取金黄色葡萄球菌的Nuc基因和大肠杆菌16S-23SrRNA基因作为扩增靶基因序列,设计两对特异性引物。通过正交试验优化反应条件建立二重PCR检测体系,优化结果表明二重PCR扩增出两条特异性条带,大小与实验设计的249bp(金黄色葡萄球菌)和375bp(大肠杆菌)相符,而对其它奶牛乳房炎主要病原菌的PCR扩增结果均为阴性;敏感性测定结果表明,该二重PCR技术能同时检出14.6pg的金黄色葡萄球菌模板和26.0pg的大肠杆菌模板。使用该二重PCR检测29份临床奶样,发现其中17份携带相关基因,并分离得到相关致病菌。     

3种条件性致病菌三重PCR检测方法的建立及初步应用

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.     

链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌多重PCR检测方法的建立及应用

根据NCBI上已收录的链球菌ef-tu基因、金黄色葡萄球菌nuc基因、沙门菌hut基因和大肠杆菌23SrRNA基因的序列,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立了能够同时检测4种细菌混合感染的多重PCR诊断方法。特异性分析结果表明,应用该方法可以从链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌以及4种细菌的混合物中扩增出4条大小分别为197、278、495和652bp的特异性条带,其他对照组的检测结果均为阴性;敏感性分析表明,该方法对4种病原菌基因组DNA的检出量分别为链球菌25.6pg、金黄色葡萄球菌33.2pg、沙门菌35.7pg、大肠杆菌52.1pg;人工模拟感染样本检测表明,该方法能从混合感染的病料中特异地检测出4种病原菌。本试验建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效的检测链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌的混合感染。     

犬细小病毒和犬冠状病毒双重PCR方法的建立及应用

根据GenBank中犬细小病毒(CPV)和犬冠状病毒(CCV)基因序列各设计了一对引物,经试验条件的优化,建立了检测CPV的PCR方法和CCV的RT-PCR方法,扩增目的片段大小分别为337 bp和852 bp.在此基础上建立了检测CPV和CCV双重PCR方法.该双重PCR方法能特异的扩增CPV和CCV,且分别扩增出1...     

Multiplex PCR assay for the detection of high virulence rabbit Staphylococcus aureus strains

High virulence rabbit Staphylococcus aureus strains, which are clonal in origin, are responsible for the spread of chronic staphylococcosis at the rabbit flock level. The aim of the present study was to develop a multiplex PCR assay that can be used for the identification of these high virulence strains. Two targets of the assay were the bbp and the selm genes, which have recently been shown to occur specifically in high virulence isolates. A third target was a sequence designated "flank", which was derived from a previously generated high virulence specific RAPD pattern. Furthermore, the femA gene, which is specific for S. aureus, was incorporated in order to avoid false negative results due to insufficient DNA preparation. The multiplex PCR was successful at differentiating the 26 typical high virulence and 50 low virulence rabbit S. aureus strains incorporated in the present study. Therefore it is useful for the initial screening of newly acquired breeding stock, in order to prevent the intake of high virulence strains in rabbitries.     

Multiplex PCR and RPLA Identification of Staphylococcus aureus enterotoxigenic strains from bulk tank milk

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.     

Real—time PCR和PCR方法快速检测犬细小病毒

为适应出入境口岸对进出境宠物快速检疫的需要,本研究在建立PCR方法检测犬细小病毒(CPV)的基础上,进一步采用Taqman探针技术建立了快速检测CPV的Real-time PCR方法.通过灵敏度对比试验,证实Real-time PCR方法比PCR方法检测灵敏度显著提高.通过对大量不同采样部位样品的检测证实,本研究建立的Real-time PCR和PCR方法具有较高的可靠性,并可显著提高CPV的阳性检出率.     

MEV、ADV和CAV三种病毒多重PCR检测方法的建立

建立一种同时检测貂肠炎病毒(MEV)、貂阿留申病毒(ADV)和犬腺病毒(CAV)的多重PCR诊断方法.引用已有的CAV引物,并根据GenBank发表的MEV、ADV序列保守区域设计特异性引物进行PCR扩增,可同时得到扩增长度为795(MEV)、451(ADV)、1 019 bp(CAV)3奈特异性片段,对猪细小病毒(PPV),犬瘟热病毒(CDV)进行PCR检测结果为阴性.各种模板、引物之间相互不构成干扰.敏感性试验证明,可以检测到模板中MEV 101.5 TCID50和CAV 100.5 TCID50的病毒含量,对ADV检测的敏感性更高.     

金色葡萄球菌、伤寒沙门氏菌和副溶血弧菌单管多重PCR检测方法的建立及初步应用

本研究根据金黄色葡萄球菌（Staphylococcus auretls，SA）耐热核酸酶（nut）基因、伤寒沙门氏菌（Salmonella typhi，ST）鞭毛抗原（H1-d）基因和副溶血性弧菌（Vibrio parahaemolyticus，VP）热稳定直接溶血素（tdh）基因，分别设计了三对引物Nuc-F／Nu-R、H1-d—F／H1-d—R和Tdh-F／Tdh-R，预计PCR扩增的DNA片断分别为279bp、202bp和458bp。通过对单个基因PCR和单管多重PCR扩增的特异性、敏感性分析以及对单管多重PCR扩增条件如引物浓度、退火温度和dNTP浓度等的优化，建立了快速检测金黄色葡萄球菌、伤寒沙门氏菌和副溶血弧菌的单管多重PCR方法，该方法检测的敏感性分别为：47．8PgST的基因组DNA．22．7PgSA的基因组DNA和0．3745PgVP的基因组DNA。模拟试验显示最低检测限度为分别为：SA150CFU／反应体系，ST98CFU／反应体系，VP53CFU／反应体系。表明该方法具有很好的应用价值和开发前景。     

绵羊嗜皮菌病PCR诊断方法的建立

为建立动物嗜皮菌病的PCR检测方法,根据GenBank发表的刚果嗜皮菌属的部分16s-rRNA基因序列,设计了1对特异性引物.经PCR扩增,从发病绵羊皮肤病料提取的基因组DNA得到了大小约500 bp的DNA片段.将PCR产物克隆并测序表明,与GenBank发表的刚果嗜皮菌基因序列的同源性为90.632%.而猪胸膜肺麦放线杆菌、大肠杆菌、葡萄球菌、肠球菌扩增结果为阴性;PCR的敏感性试验显示,这时引物能够检测到1 ng的DNA.表明此PCR方法特异性好,敏感性高,可用于嗜皮菌病的快速诊断.     

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk

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.