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1.
副猪嗜血杆菌和猪链球菌双重PCR方法的建立与应用   总被引:1,自引:0,他引:1  
针对副猪嗜血杆菌和猪链球菌16S rRNA序列各设计1对特异性引物,分别能扩增出822 bp和294 bp的DNA片段,并据此建立了快速准确鉴别副猪嗜血杆菌和猪链球菌的双重PCR方法,临床试验证明该方法具有很好的特异性、敏感性,能对临床病料进行鉴别诊断.对161份临床样本检测结果显示,副猪嗜血杆菌的检出率为23.75%,猪链球菌的检出率为43.48%,二者混合感染率为9.94%.  相似文献   

2.
猪传染性胸膜肺炎PCR诊断方法的建立   总被引:12,自引:2,他引:10  
根据已发表的猪胸膜肺炎放线杆菌APXIV毒素的基因序列,自行设计和合成了二对可扩增448bp和365bp目的片段的引物,成功的建立了检测APP的套式PCR方法。通过对猪肺疫巴氏杆菌、猪链球菌、大肠杆菌、猪嗜血杆菌、猪肺炎支原体和猪丹毒杆菌的DNA进行了PCR检测,结果均为阴性;对猪胸膜肺炎放线杆菌的1、2、5、6、7、9国际标准血清型均扩增出448bp和365bp的特异性条带;检测的敏感度一步PCR可达到5OO个细菌,最低检出DNA浓度可达到0.585ng/mL;套式PCR可达到50个细菌,最低检出DNA浓度可达到58.5pg/mL。另外,对5株从病猪体内分离的猪胸膜肺炎放线杆菌进行了检测,5株均成阳性反应;对10只屠宰猪的肺脏分离物进行了检测,结果1份为阳性。结果表明此法特异性和敏感性均很高,可做为猪传染性胸膜肺炎的快速诊断和流行病学调查的手段。  相似文献   

3.
新城疫病毒强弱毒株PCR-RFLP鉴别方法的建立   总被引:3,自引:0,他引:3  
设计1对特异性引物扩增新城疫病毒F蛋白基因460 bp大小片段,而后利用PstⅠ和Bbe Ⅰ进行酶切,弱毒株酶切出120 bp和220 bp两个片段,而强毒株酶切出120 bp和340 bp两个片段,经敏感性、特异性试验,表明该方法敏感性高、特异强.用该PCR-RFLP检测了170份病料,检出强毒株5份、弱毒株1份,PCR阳性病料经SPF鸡胚进行病毒分离,用MDT、ICPI试验测定其生物学毒力,并对它们的F蛋白基因克隆测序,通过MDT、ICPI和推导氨基酸裂解位点分析,确定其毒力的结果与PCR-RFLP检测结果一致.表明该方法快速、特异、敏感,很适合于鸡群中新城疫快速诊断和新城疫病原学监测.  相似文献   

4.
猪链球菌2型的PCR快速检测   总被引:37,自引:4,他引:33  
根据猪链球菌 2型的荚膜多糖抗原基因 cps2 J,合成 1对可扩增长度为 6 75 bp目的片段的引物 ,建立了检测猪链球菌 2型的 PCR法。应用 PCR对 9株经玻片凝集试验检测为猪链球菌 2型的菌株进行了检测 ,均呈阳性 ;而对马链球菌兽疫亚种 (C群 )、猪葡萄球菌、猪丹毒杆菌、猪肺疫巴氏杆菌、猪肺炎霉形体等检测结果均呈阴性 ,表明了本方法的特异性。用此法对 88份正常猪的扁桃体样品的细菌分离物进行了检测 ,36份呈阳性 ,同时用玻片凝集试验进行对照检测 ,也全部呈阳性。而此法不需进行细菌的纯分离培养 ,即可用于猪链球菌 2型的快速诊断以及流行病学调查。  相似文献   

5.
逆转录—聚合酶链反应(RT—PCR)检测轮状病毒   总被引:11,自引:1,他引:10  
为了给轮状病毒感染的诊断和流行病学研究提供更为敏感和可靠的手段,选取A组轮状病毒VP7基因上的2段高度保守序列作为引物,在优化逆转录-聚合酶链反应(RT-PCR)条件的基础上,建立了检测轮状病毒的RT-PCR方法。通过对比试验,确定了PCR的最优模式:94℃变性1min→55℃退火1min→72℃延伸2min,30个循环后再在72℃下延伸10min。用此模式进行了RT-PCR的特异性和敏感性试验。检测的6株轮状病毒分离株(牛HN-7、BRV007、BRV014、BRV6555、猪Li99、Nan86)及2株参考株(牛NCDV、猴SA11),都能扩增出唯一的342bp的目的条带;对猪流行性腹泻病毒(PEDV)及传染性胃肠炎病毒(TGEV)感染猪的粪样、正常MA104细胞检测结果均呈阴性;检测的敏感度可达1pg水平。对40份猪、牛、兔的腹泻粪样检测,30份呈阳性,而用作平行对照的夹心ELISA法检测,有25份呈阳性,两者符合率为87.5%。两法检测不符的5份粪样的PCR扩增产物,用地高辛标记探针进行了斑点杂交,结果均呈阳性,表明RT-PCR法比ELISA法敏感性高。  相似文献   

6.
猪瘟病毒石门株E2基因的RT-PCR克隆及鉴定   总被引:2,自引:0,他引:2  
本文采用RT-PCR技术从感染猪血中成功扩增了我国猪瘟病毒强毒石门株E2基因,大小为1184bp,与预期大小一致。经巢式PCR和酶切鉴定证实所扩增的片段为E2基因特异性片段。将扩增的E2基因克隆到P^GEM-T载体,采用限制性内切酶鉴定和PCR技术了阳性重组子。  相似文献   

7.
猪细小病毒PCR检测方法的建立与应用   总被引:21,自引:0,他引:21  
根据已报道的猪细小病毒基因组序列,设计并合成了1对寡核苷酸引物,通过对影响PCR扩增因素的筛选,成功地从猪细小病毒感染的组织和细胞中扩增出445bp片段,回收该片段用EcoR I酶切得到了预期的结果,证实了该扩增片段的特异性。敏感性试验表明,该方法可以检测出10^-4个TCID50的病毒含量。利用该方法对临床上24份流产病科的检测,查出阳性16份,而同时利用HA检测阳性只有10份。这些结果说明本试验建立的PCR诊断方法灵敏度高、特异性强。  相似文献   

8.
用聚合酶链反应(PCR)和限制性核酸内切酶分析(REA)对牛精液中胎儿弈杆菌进行快速筛选,是对传统增减分离法的有效补充。选择胎儿弯杆菌16SrRNA高变区寡核苷酸配对引物(C1/C2)用PCR扩增362bp片段。完成一次PCRT/REA过程需10小时,对实验感染公牛的精液及用牛或蛋黄--Tris(EYT)稀释的业液的检测敏感性为3个胎儿弯曲杆菌性病亚种菌体。本试验对胎儿弯杆菌两个亚种及其它一些弯杆  相似文献   

9.
肺炎克雷伯菌Ⅲ型菌毛PCR检测方法的建立   总被引:1,自引:0,他引:1  
以肺炎克雷伯菌Ⅲ型菌毛主要结构亚单位基因为靶序列,设计合成了1对可扩增长609 bp目的片段的引物,建立检测肺炎克雷伯菌Ⅲ型菌毛结构基因的PcR方法.特异性试验表明,本室保存的表达Ⅲ型菌毛的鸡源肺炎克雷伯菌临床分离株Kpn7扩增出609 bp的特征性片段,而大肠埃希菌、副伤寒沙门菌、胸膜肺炎放线杆菌、猪链球菌和金黄色葡萄球菌扩增产物均未检出;敏感性试验表明,PER方法检测该菌基因组DNA的灵敏度为16.8 ng/L,检测该菌菌液的灵敏度为3.7× 102 CFU/mL.用此方法对10份临床疑似病例分离物进行检测,结果有5株阳性.表明此方法特异性和敏感性都很高,可用于临床Ⅲ型菌毛肺炎克雷伯菌病的辅助诊断和流行病学调查.  相似文献   

10.
猪胸膜肺炎放线杆菌PCR诊断方法的建立与应用   总被引:5,自引:3,他引:5  
根据胸膜肺炎放线杆菌ApxⅣ基因设计1对引物,扩增特异的650bp棱酸片段,建立了应用PCR检测猪胸膜肺炎放线杆菌的方法。特异性试验结果表明,12个血清型的放线杆菌参考菌株均能扩增出650bp特异性的核酸片段,而大肠杆菌、多杀性巴氏杆菌、猪肺炎支原体、伤寒沙门氏菌和支气管败血性波氏杆菌的扩增结果均为阴性。敏感性试验结果表明,PCR的最低检出限量为500个放线杆菌。利用建立的PCR检测方法对22株从山东省不同地区分离的疑似胸膜肺炎放线杆菌菌株进行检测.结果14株为阳性。对感染猪病变组织的检测结果表明,病变部位不同,胸膜肺炎放线杆菌的检出率不同,其中以扁桃体的检出率最高。  相似文献   

11.
We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP+EF+) and highly virulent S. suis type 1 strains (MRP(s)EF+) and of the EF protein of weakly virulent S. suis type 2 strains (MRP+EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.  相似文献   

12.
猪链球菌2型及其毒力因子检测多重PCR的建立与应用   总被引:8,自引:3,他引:8  
根据相关文献设计并合成引物,建立了能同时检测猪链球菌2型(cps)及其重要毒力因子溶菌酶释放蛋白(mrp)和细胞外因子(epf)的多重PCR方法。目的片段的大小分别为885bp(mrp)、675bp(cps)和443bp(epf)。对参考菌株、人工攻毒病料和临床收集病料的检测结果显示,该多重PCR特异性强、敏感性高,可直接从临床病料中检测出猪链球菌2型,并能鉴定其毒力因子表型。  相似文献   

13.
猪链球菌2型多重PCR检测方法的建立及应用   总被引:1,自引:0,他引:1  
设计3对引物,分别扩增链球菌属特异性gdh、猪链球菌种特异性16S rRNA和猪链球菌2型特异性cps2J等基因,目的片段大小分别为725bp、523bp和387bp。利用合成的3对引物并通过对反应条件与反应体系的优化建立了多重PCR。应用该多重PCR检测了分离到的链球菌1105株,检出猪链球菌667株,猪链球菌2型33株。研究结果表明,该方法特异性高、敏感性强,可广泛应用于猪链球菌病的快速诊断及流行病学调查。  相似文献   

14.
四川猪2型链球菌病的PCR检测   总被引:4,自引:0,他引:4  
2005年7月中下旬,四川贵阳等地人、猪陆续发生一种发病惠、死亡快、高度散发的疾病,从病死猪组织中分离出19株链球菌,设计合成3对引物,分别对分离菌株进行了猪链球菌16SrRNA、CPS2J、MRP基因片段扩增,有15株菌3个基因的扩增结果均为阳性,表明此次疲情的病原为猪2型链球菌。  相似文献   

15.
根据猪链球菌2型荚膜多糖和马链球菌兽疫亚种类M蛋白的保守区序列分别设计了2对简并引物,建立了一种能同时检测猪链球菌2型和马链球菌兽疫亚种的双重PCR方法。结果显示,该双重PCR能从100个细菌的混合纯培养物中扩增出2条目的片段。而且可以直接从病料组织中检测到相应的病原菌。用建立的双重PCR方法和细菌分离培养法平行检测人工感染的组织病料,PCR方法与细菌培养法的阳性检出率基本一致,但PCR方法的特异性好、敏感性高,简便易行,可以用于猪链球菌病的流行病学调查和实验室的快速鉴别诊断。  相似文献   

16.
猪链球菌2型对PK-15细胞的黏附动力学   总被引:1,自引:1,他引:0  
为研究携带不同毒力因子的猪链球菌2型对细胞侵袭作用的差异,本实验室从全国各地分离100多株猪链球菌,PCR鉴定出10株链球菌2型,其中携带毒力因子菌株为7-4[MRP+EF+]、8-3[MRP+EF-]、ZH-1[MRP-EF+],另外选择1株LN-2 [MRP-EF-]与PK 15细胞进行黏附动力学试验,结果表明MPR毒力因子与菌株对PK-15细胞黏附具有密切相关性。  相似文献   

17.
从送检的一份疑似链球菌病病死猪病料中分离出1株细菌,通过对分离菌株进行细菌的培养特性、生化试验、玻片凝集试验及PCR分型鉴定,确定为2型猪链球菌,动物致病性试验表明,1×108 CFU/mL的剂量可致死小白鼠,对该分离菌株的毒力因子进行PCR检测发现,溶菌酶释放蛋白(mrp)、胞外因子(ef)和溶血素(sly)均为阳性。此菌的分离鉴定为自家灭活菌苗的制备提供了候选菌株。  相似文献   

18.
Virulence-associated gene profiling of Streptococcus suis isolates by PCR   总被引:11,自引:0,他引:11  
Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.  相似文献   

19.
A total of 323 isolates of Streptococcus suis recovered from diseased or healthy pigs in France were serotyped. The presence of virulence-related proteins, Muraminidase-Released Protein (MRP), Extracellular Factor (EF) and Suilysin was also studied in 122 isolates of capsular types 2, 1/2, 9, 7 and 3 to evaluate their implication in virulence of S. suis. Capsular types 2, 1/2, 9, 7 and 3 were the most frequently detected (93%), with 69% for the capsular type 2 alone. Capsular types 2, 1/2, 9, 7, 3, 1, 4, 8, 18, 10 and 12 were isolated from diseased pigs, whereas types 2, 7, 9, 1/2, and 3 originated from the nasal cavities or tonsils of healthy animals. Most of the S. suis type 2 isolates recovered from diseased pigs carried MRP+ EF- Suilysin- (46%) or MRP+ EF+ Suilysin+ (28%) phenotypes. The MRP+ EF- Suilysin- phenotype was also detected in 67% of S. suis type 2 strains isolated from healthy pigs. The production of the virulence-related proteins was less frequently found in S. suis types 1/2, 9, 7 and 3 recovered either from diseased or healthy pigs. In this study, all the capsular type 1/2 strains were MRP+ EF- Suilysin- and all the S. suis type 7 harboured an MRP- EF- Suilysin- phenotype. The MRP- EF- Suilysin- phenotype was found in S. suis types 2, 3, 7 and 9 isolated from septicaemia, meningitis, pneumonia, and pleurisy. These results suggest that the presence of these proteins should not be used as a single condition for classifying the virulence of a field isolate in France.  相似文献   

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