不同来源沙门菌入侵基因invH的检测及序列分析

根据沙门菌invH基因序列设计1对引物,对9株沙门菌和4株常见病原菌进行PCR扩增,并将扩增出的in-vH基因进行克隆及序列分析,应用生物信息学方法预测invH蛋白的T细胞抗原表位。结果表明,9株沙门菌PCR均扩增出519bp的特异条带,4株非沙门菌均无特异带扩增;核苷酸序列分析证实,3株鼠伤寒沙门菌之间同源性为99.8%,与鸡白痢和鸡肠炎沙门菌之间的核苷酸序列同源性为98.6%,3株猪伤寒沙门菌和3株鸡源性沙门菌同源性较低,且不在同一支系统进化树上;invH蛋白有5个重复率最高的T淋巴细胞受体识别的抗原表位。     

鸡白痢与鸡伤寒沙门菌双重PCR方法的建立和初步应用

分析不同血清型/生物型沙门菌全基因组序列筛选出鸡白痢和鸡伤寒沙门菌特异性基因组序列,设计两对引物,建立双重PCR方法鉴别检测鸡白痢和鸡伤寒沙门菌不同生物型,并进行初步的临床应用。双重PCR方法结果显示,鸡白痢沙门菌显示417 bp条带,鸡伤寒沙门菌显示417 bp和636 bp两个条带,而阴性对照未出现条带,与预期设计相符。双重PCR体系对56株不同血清型沙门菌鉴定结果与细菌学分离的血清型鉴定结果完全一致,说明本试验建立的双重PCR体系特异性良好,应用上述方法检测鸡场疑似20份临床样本,结果发现8株鸡白痢沙门菌阳性,1株鸡伤寒沙门菌阳性。上述结果表明,已建立双重PCR方法特异性检测鸡白痢和鸡伤寒沙门菌。本试验为鸡白痢和鸡伤寒不同生物型沙门菌的检测提供了一种简洁、敏感、特异的新方法。     

辽宁省规模化猪场生产链中沙门菌优势血清型及其主要毒力因子分布

为了解辽宁省猪沙门菌流行菌株主要血清型毒力因子的分布特征及致病性,依据猪沙门菌属保守基因、多重PCR引物和血清凝集试验,从辽宁省选择有代表性的规模化养猪场、下游生猪屠宰场和肉类市场采集肠道、饲料和污水、猪肉等样品进行猪沙门菌及其血清型鉴定。应用猪沙门菌14种主要毒力因子特异性基因扩增检测方法,检测所分离到的不同血清类型猪链球菌的毒力因子分布情况,并应用小鼠攻毒试验和病理学技术对其致病性进行观察。结果显示:采集的412个样品中共分离到39株猪沙门菌,养猪场、屠宰场、销售市场所分离沙门菌检出率分别为51.28%,41.02%,7.70%;主要血清型为猪霍乱沙门菌、肠炎沙门菌、鼠伤寒型沙门菌,其阳性率分别为41.02%,38.46%,5.12%,未定型占15.38%;毒力因子共检出11种,其中检出率达到90%以上的和3种血清型共同携带的毒力因子结果一致有mogA、sseL、mgtC、bcfA、araB、stn,猪生产链3个环节共同携带的毒力因子有spvR、spvA、mogA、mgtC、araB、stn,其中养殖场毒力因子检出率最高,销售市场毒力因子检出率最低;猪霍乱沙门菌可引起小鼠的急性败血症,肠炎沙门菌和鼠伤寒沙门菌均可引起小鼠发病,但各器官的损伤则较轻。     

徐州及周边地区鸭源沙门菌的分离鉴定及其耐药性、毒力基因分析

旨在了解徐州及周边地区鸭源沙门菌血清分布、耐药状况及毒力基因携带情况。采集徐州及周边地区病鸭脏器329份进行沙门菌分离、双重PCR鉴定及血清分型;采用Kirby-Bauer(K-B)法进行抗菌药物敏感性试验;设计10对引物,对6大类耐药基因进行检测;PCR检测与沙门菌致病性相关的8个毒力基因。结果:共分离49株鸭源沙门菌,分离率为14.9%;鉴定出13种血清型,优势血清型为印第安纳沙门菌、阿贡纳沙门菌和鸭沙门菌。耐药性结果显示:分离株对氨苄青霉素(AMP)和头孢曲松(CRO)耐药率为100%,对土霉素(OXY)、阿莫西林/克拉维酸(AMC)、卡那霉素(KAN)、利高霉素(NA)、强力霉素(DOX)、恩诺沙星(ENR)、环丙沙星(OFL)抗菌药物的耐药率均超过50%以上。耐药基因blaTEM-1检出率为100%,blaPSE-1、aadA1、aadA2、tetA、gyrA、sul2基因检出率超过50%以上,parC、floR和clmA基因检出率分别为44.8%、30.6%和26.5%。毒力基因检测发现spvR(12.2%)携带率较低,mgtC和stn基因检出率为100%,invJ、sseL、siiE、sopB以及fimA基因携带率分别为75.6%、53.3%、89.6%、98.2%和97.9%。研究结果表明,徐州及周边地区鸭沙门菌血清型分布较广,分离株的耐药性及多重耐药较严重,耐药基因和毒力基因分布广泛。     

动物粪便沙门菌PCR检测技术的研究

为了建立一种动物粪便样品中快速、特异、敏感的沙门菌检测方法,根据沙门菌肠毒素stn基因设计一对引物,对不同血清型的沙门菌和非沙门菌进行PCR检测,并对该PCR方法进行反应的灵敏度、模拟粪便样品的最低检出限测定及粪便样品的检测。运用该方法对250份粪便样品进行检测,同时用传统方法进行验证。结果表明,设计的引物特异性好,能专一性扩增出约260 bp条带;该引物灵敏度高,能进行有效检测的核酸最低起始量为43.85 pg,纯菌检测灵敏度达1 cfu/mL。接种沙门菌到粪便样品中,当粪便样本中菌量达103cfu/mL以上时,不需要增菌,沙门菌可立即检出,增菌后检出限可以达到1 cfu/mL。PCR检测250份样品共检出18份阳性,同时传统细菌培养方法证明结果正确。该方法可用于粪便样品的检测。     

禽源沙门菌多重PCR检测方法的建立与应用

为了快速检测禽源沙门菌,根据已知的沙门菌全基因组序列,通过分析筛选出肠炎沙门菌、鸡伤寒沙门菌和鼠伤寒沙门菌三种血清型的特异保守序列,设计PCR引物,建立多重PCR检测方法。结果表明,该方法可以特异性检测肠炎沙门菌、鸡伤寒沙门菌和鼠伤寒沙门菌,检测灵敏度分别为6.413、4.363和8.724 pg/反应。临床样品检测结果表明,多重PCR方法的检测灵敏度(54/445)高于传统细菌学检测方法(33/445)。     

猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxIICA基因的克隆和基因缺失

猪传染性胸膜肺炎放线杆菌血清型7型25＿4株ApxIICA基因用特异性引物进行PCR扩增并克隆到T载体上,构建重组质粒pMD18＿ApxIICA,将pMD18＿ApxIICA转化到E.coliJM83中并测序。序列分析表明血清型7型25＿4株ApxIICA与其它血清型(5型和9型)核苷酸序列同源性达98%以上,氨基酸序列同源性达90%以上。利用ApxIIC基因内部单一的SpeI位点,设计特异性的引物,利用PCR技术在ApxIIC基因内部缺失165bp的核苷酸片段,PCR产物再用SpeI进行酶切,并体外连接构建得到含有ApxIIC缺失基因的重组质粒pMD18＿ApxII△CA。     

猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxⅡCA基因的克隆和基因缺失

猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxIICA基因用特异性引物进行PCR扩增并克隆到T载体上,构建重组质粒pMD18-ApxIICA,将pMD18-ApxIICA转化到E.coli JM83中并测序.序列分析表明血清型7型25-4株ApxII CA与其它血清型(5型和9型)核苷酸序列同源性达98%以上,氨基酸序列同源性达90%以上.利用ApxIIC基因内部单一的SpeI位点,设计特异性的引物,利用PCR技术在ApxIIC基因内部缺失165bp的核苷酸片段,PCR产物再用SpeI进行酶切,并体外连接构建得到含有ApxIIC缺失基因的重组质粒pMD18-ApxII△/CA.     

猪链球菌谷氨酸脱氢酶基因的克隆和序列分析

对猪链球菌2型、7型、9型的标准菌株和7株猪链球菌2型分离菌的谷氨酸脱氢酶(GDH)基因进行PCR扩增,经回收纯化后克隆到PMD18-T载体,筛选阳性克隆菌后测序并对其进行序列分析。结果,PCR扩增出1300bp左右的片段,包括GDH基因的整个开放阅读框,而测序结果表明,其序列与GenBank中的猪链球菌GDH基因序列一致,进一步序列分析表明,GDH的核苷酸序列在相同血清型之间同源性高达99%以上,而在不同血清型之间的同源型也达到了96%以上,而其氨基酸序列同源性则都在99%以上,且都具有GDH1型家族的功能区域。说明猪链球菌GDH基因及其蛋白具有高度的保守性,为进一步研究与应用提供了重要依据。     

鸭疫里氏杆菌PCR方法初步建立及应用

为了建立针对鸭疫里氏杆菌的病原学检测方法,试验根据已发表的鸭疫里氏杆菌的外膜蛋白A(OmpA)的基因序列(GenBank登录号为AF104937)设计1对引物,对7株不同血清型鸭疫里氏杆菌进行扩增并建立PCR方法.结果表明:均扩增出与预计大小一致的目的片段,PCR方法敏感性可达到8.6 Pg;而多杀性巴氏杆菌、沙门菌、...     

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.     

The detection and determination of potential virulence of Salmonella spp. using PCR method

The aim of this study was to prove that PCR is a very useful method to identify Salmonella strains and to determine their virulence factors by amplification of characteristic genetic markers. Investigations included 5 strains of Salmonella which were obtained from pure cultures and 1 Salmonella strain from the mixed culture. Genotypic analysis of 6 examined strains revealed the 163-bp fragment of chromosomal DNA, which is the DNA rep. ori. gene, encoding the particular genus. In all of these strains 215-bp, 203-bp and 204-bp chromosomal DNA fragments were identified as representing the stn, stpA and spaO genes that confirmed their virulence. These amplification products were identified in both pure and mixed culture from pork. Sensitive and rapid PCR method may be used not only for identification of Salmonella strains and for determination of their virulence factors but also for routine microbiological diagnostic of food pathogens.     

Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection.

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.     

An allele-specific PCR assay for the rapid and serotype-specific detection of Salmonella pullorum

Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.     

Isolation,Identification and Pathogenicity of Salmonella typhimurium from Guizhou Pony

To investigate the reason of the diarrhea in Guizhou pony,we used the feces of pony as experimental material to isolate and detect pathogenic bacteria.S6 strain was isolated from SS and XLD medium,and identified using Gram staining,biochemical tests and molecular phylogeny methods.The results showed that S6 strain could growth on SS and XLD medium,and the Gram staining was negative.Biochemical test suggested that its phenotype features were accordance with Salmonella.The 16S rRNA gene sequence of S6 strain was determined in a nucleotide sequence identity of 99% with Salmonella typhimurium ATCC 13311 (NR_119108).Based on the morphological and molecular phylogenetic results,the strain was identified as Salmonella typhimurium S6 strain.It was virulent to mice with the median lethal dose (LD₅₀) of 4.71×10² CFU.Then,we amplified the invasion protein A (invA) gene by PCR method.The invA gene isolated from S6 strain contained 6 to 8 bp different from the known gene,which resulted in only one amino acid substitution.The mutant sites of invA gene might attribute to the pathogenicity of S6 strain.The detection rate of Salmonella was 57% in Guizhou pony population.It was inferred that the diarrhea in Guizhou pony might be caused by virulent Salmonella typhimurium.     

Establishment of the PCR system specific to Salmonella spp. and its application for the inspection of food and fecal samples

We established the PCR detection system specific to Salmonella species using Salmonella enterotoxin gene (stn). The detection limit was one bacterial cell per one gram of fecal and minced-meat samples using enrichment procedure by Tripticase soy broth or Salmonella enrichment broth, respectively. We concluded that this PCR system is useful for the practical application in the field of the public hygiene.     

大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体构建

合成胸膜肺炎放线杆菌质粒pKMA2425上长度为126bp及2214bp的2个DNA片段,克隆到大肠埃希氏菌质粒pGSI中,获得重组质粒pGSIA。PCR扩增pTKRED上的大观霉素抗性基因及线性化的pGSIA（126bpDNA片段-pGSI-2214bpDNA片段）,连接,电转化胸膜肺炎放线杆菌,在大观霉素的选择压力下筛选鉴定到连接正确的质粒,命名为pGSIAS。pGSIAS测序结果符合预期。在大观霉素的选择压力下,含有pGSIAS的胸膜肺炎放线杆菌血清1-10型菌株均生长良好;在氨苄青霉素的选择压力下,含有pGSIAS的大肠埃希氏菌生长良好。结果表明本研究构建的大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体pGSIAS序列正确,在胸膜肺炎放线杆菌及大肠埃希氏菌中均能复制并表达质粒上的相关基因。     

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction

A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.     

贵州矮马鼠伤寒沙门氏菌的分离鉴定及其致病力研究

为了探究贵州矮马的腹泻病原,试验以腹泻粪样为材料进行病原菌的分离鉴定。经SS和XLD培养基筛选,得到1株菌株S6,经革兰氏染色、生理生化检验以及16S rRNA基因序列比对分析,S6菌株可在SS和XLD琼脂培养基表面生长,革兰氏染色呈阴性;生理生化检验结果与沙门氏菌的生化特性相符;16S rRNA基因序列与已知的鼠伤寒沙门氏菌ATCC 13311（登录号：NR_119108）的亲缘关系最近,序列同源性为99％。形态学和分子系统学分析表明,该菌株为鼠伤寒沙门氏菌。采用PCR方法从S6菌株基因组中得到侵袭蛋白A（invasion protein A,invA）基因,与已知基因序列有6~8 bp不一致,编码的氨基酸发生了一个替换。通过改良寇氏法测定S6菌株对小鼠的半数致死量（LD₅₀）为4.71×10² CFU,属于强致病力菌株。推测S6 invA基因的变异可能与菌株的毒力强弱有关。贵州矮马群体中沙门氏菌的检出率为57％。本研究提示贵州矮马的腹泻病原之一可能是强毒力的鼠伤寒沙门氏菌。