COMPETITIVE ENZYME IMMUNOASSAY FOR THE RAPID IDENTIFICATION OF SALMONELLA

A competitive enzyme immunoassay using a murine monoclonal antibody M105 directed against a genus-specific epitope in the Salmonella lipopolysaccharide was used to identify over 200 strains of Salmonella submitted to the National Laboratory for Enteric Pathogens. The immunoassay rapidly identified 208 strains of Salmonella representative of subspecies I, II, III_a, III_b, IV, and V, including 89 different serotypes from 26 O serogroups. The competitive enzyme immunoassay did not give positive results with 3 strains of Citrobacter freundii and 4 strains of Escherichia coli which were submitted to our laboratory as suspect Salmonella.     

鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC~I的克隆和鉴定

以提纯的鼠伤寒沙门氏菌8705染色体DNA为材料,经EcoR Ⅰ消化,过SepharcylS-400柱,得到大于400bp的酶切片段;然后随机克隆到质粒pGEM-3Zf(—)中,转化大肠杆菌LC2a(hag~-,recA~-);在氨苄青霉素平板上共得到6013个转化子,从中筛选出1个有动力的克隆,小量制备质粒DNA,经酶切电泳鉴定,该克隆的外源片段大小为15.3kb,将其命名为pGI4015。动力、动力抑制试验和Southern blot分子杂交试验证明pGI4015中载有鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC~I;利用其BamH Ⅰ和Sal Ⅰ位点删除与鞭毛蛋白表达无关的序列,构建亚克隆质粒pGI4015BS,使得fliC~I定位于更小的区域——3.8kb的BamH Ⅰ/Sal Ⅰ插入片段上。     

MULTIPLEX DETECTION OF ESCHERICHIA COLI AND SALMONELLA ENTERITIDIS BY USING QUANTUM DOT-LABELED ANTIBODIES

In this study, we demonstrated the simultaneous detection of Escherichia coli and Salmonella enteritidis, by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti- E. coli and anti- Salmonella antibodies. QD–antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD–antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead–bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 × 10² to 5 × 10⁵ cfu/mL for E. coli and 4  ×  10² to 4  ×  10⁵ cfu/mL for S. enteritidis .

PRACTICAL APPLICATIONS

In this study, antibody-conjugated multicolor quantum dots (QDs) were used for simultaneous detection of Escherichia coli and Salmonella enteritidis . The results of this study indicate that QD labels can be used in multiplex, rapid and selective detection of bacteria with detection limits comparable with those of many novel methods in cases where the assay conditions are optimized. Furthermore, the assay can be modified for the simultaneous detection of more than two species through using QD labels having different emission wavelengths.     

RAPID PURIFICATION OF SALMONELLA DNA IN MINCED MEAT AND DETECTION BY REAL-TIME PCR

Four rapid and simple DNA purification and sample treatment protocols were evaluated for detection of Salmonella enterica in spiked minced meat, using a fluorogenic 5'nuclease (TaqMan) PCR assay in an ABI-Prism 7700 Sequence Detector. The detection limit with the single separation treatment of DNeasy was found to be 6–8 CFU in just 19 end-point fluorescence (C_t) values, while this was 22 C_t for a combination of DNeasy and BactXtractor. Extraction by DNeasy resulted in C_t <22, indicating a detection limit of <10 Salmonella cells per 25g, when the samples were inoculated with Salmonella before the overnight preenrichment. The method is currently being adapted to a BioRobot 3000 platform. However, the use of paramagnetic beads (DNA Direct) resulted in poor and variable detection limit.     

微菌落观察法快速检测和鉴别结核杆菌培养物的研究

将取自肺结核患者的219例痰标本接种匡氏琼脂平板,共分离到112例结核菌生长物。其中培养法104例(92.8％)阳性,微菌落观察法108例(96.4％)阳性,微菌落法阳性率稍高。培养法和微菌落法阳性标本首次检出时间分别为18.6d和11d,微菌落检出时间更短(P<0.01)。常规菌型鉴别方法与微菌落法对结核杆菌菌型鉴别的符合率为99％。     

RAPD分析快速鉴定双歧杆菌

本文应用ＲＡＰＤ技术,选用１１种引物,以嗜酸乳杆菌为对照,对６种１３珠双歧杆菌菌株基因组ＤＮＡ进行ＰＣＲ扩增,分析其ＤＮＡ指纹图谱,并计算其相似性指数。结果表明,双歧杆菌和非双歧杆菌之间,其相似性指数有显著差异;选择合适的引物进行扩增,双歧杆菌不同种间和同种不同株间可表现不同的ＤＮＡ指纹图谱。本文还对ＲＡＰＤ技术应用于双歧杆菌分类鉴定的可能性进行讨论。     

APPLICATION OF A TRANSPOSON FOOTPRINTING TECHNIQUE FOR RAPID IDENTIFICATION OF SALMONELLA TYPHIMURIUM TN5 MUTANTS REQUIRED FOR SURVIVAL UNDER DESICCATION STRESS CONDITIONS

Salmonella spp. are one of the foodborne pathogens that can be isolated in the environments of poultry houses and desiccation is a potential stress condition that can influence the survival of Salmonella spp. in this environment. In order to investigate the desiccation survival mechanism of Salmonella spp. the genome of S. typhimurium ATCC 14028 was screened for the genes potentially required for survival during desiccation using a novel method based on Tn5 mutagenesis previously developed in our laboratory. This method, termed transposon footprinting, simultaneously amplifies the Tn5-flanking sequences in a complex pool of the Tn5 mutants. As the length of the amplified DNA fragment should be unique for each distinct Tn5 mutant, the polymerase chain reaction (PCR) products separated on an agarose gel generate transposon footprints with each band in the footprint representing the corresponding Tn5 mutant. By comparing the transposon footprints from the pools of S. typhimurium Tn5 mutants before and after exposure to desiccation, Tn5 mutants that were not recovered after the selection were rapidly identified that would be easily isolated for further genetic analysis.     

介绍一种鉴定胚胎干细胞基因打靶快速、经济的筛选新方法

小鼠基因剔除动物模型越来越广泛地应用于哺乳动物基因功能与疾病的研究。然而每当胚胎于细胞同源重组的效率过低时,鉴定与分离带有定位变异的阳性克隆就会既费力又昂贵。本工作以类固醇受体共激活子基因为例,研究出一种快速鉴定阳性克隆的新方法。在构造重组载体时,将一段编码半乳糖苷酶的DNA序列整合到共激活子基因的蛋白起始码后面。于是,在干细胞内同源重组发生以后,半乳糖苷酶的表达就会受控于内源性共激活子基因的启动子。在载体与半乳糖苷酶DNA随机整合的大多数非特异克隆中,因为缺少启动子或由于不正确的氨基酸编码连接,导致合成牛乳糖苷酶的可能性较小。因此,在半乳糖苷酶染色阳性的克隆中,具有特异突变的阳性克隆可以富集30倍以上。从牛乳糖苷酶的阳性克隆中,再用Southern Blot方法进一步确认带有基因剔除的阳性克隆就大大减少了工作量。因为半乳糖苷酶的细胞化学染色法简便而可靠,所以在重组效率低时,可以用这种方法在短期内筛选大量克隆。但是应该注意,应用该方法的前提条件是所研究的基因必须在胚胎干细胞内表达。这种方法更为重要的意义在于当带有基因剔除的胚胎干细胞发育成小鼠后,牛乳糖苷酶的组化染色法可以轻而易举地用来揭示所研究基因在动物不同组织与细胞中的表达水平。     

利用RAPD技术快速鉴定番茄体细胞无性系变异

从以镰刀菌酸为选择剂筛选的番茄再生植株和未经筛选的植株叶片中提取ＤＮＡ,建立了适合番茄ＲＡＰＤ分析的ＰＣＲ条件,进一步在６０个随机引物中找到了４个可用于鉴定四个番茄品种体细胞无性系变异的引物。利用该法鉴定体细胞无性系变异不仅简单、迅速、可靠,而且因ＤＮＡ的用量少,不影响被检植株的后期生长,可尽早淘汰那些生理适应性而非分子水平发生变异的再生植株。     

USE OF THREE RAPID DETECTION SYSTEMS TO EVALUATE THE PREVALENCE AND DISSEMINATION OF SALMONELLA IN A BRAZILIAN POULTRY SLAUGHTERHOUSE

Prevalence and dissemination of Salmonella in a Brazilian poultry slaughterhouse were evaluated by three rapid detection systems (SS/SV^TM, VICAM, OSRT^TM, Unipath/Oxoid, and REVEAL^TM, Neogen), plus the conventional procedure. The carcasses were sampled after bleeding (P1), defeathering (P2), evisceration (P3), washing (P4), chilling (P5) and the packaged end-product (P6). In the first set of carcasses, the Salmonella incidence determined by the conventional method was 38.3% and 22.5% by SS/SV^TM. In the set for evaluation of OSRT^TM, the number of positive samples was the same detected by the cultural procedure (49.0%). In the third set, the positivity by the conventional procedure was 33.3%, and 5.0% by REVEAL^TM. The comparisons of positives in the first and third sets of carcasses were significantly different (P < 0.05). The positivity for Salmonella, in carcasses at P1 to P6, as determined by at least one of the methods, was 47.5%, 47.5%, 32.5%, 30.0%, 30.0% and 37.7%, respectively.     

PCR-BASED FLUORESCENT METHOD FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN POULTRY SAMPLES

A DNA binding fluorescence method based on polymerase chain reaction (PCR) products was evaluated for rapid detection of Salmonella Typhimurium in poultry products. Wash water samples of chicken carcasses and ground turkey were inoculated with S. Typhimurium to obtain final concentrations of 10° - 10⁵ CFU/mL. One mL of each sample was used to get the DNA template and 5 μL of the sample template was added into 25 μL of SYBR Green PCR Master Mix and two specific Salmonella ompC gene primers. The negative control was the same except 5 μL of each wash solution was added instead of 5 μL sample template. The reaction was carried out in a thermocycler. Finally, the fluorescence signal of each PCR product was measured using a fluorometer. The PCR products were also confirmed by ethidium bromide agarose gel, and the DNA concentrations of the PCR products were measured by a filter fluorescence photometer. The results showed that when bacterial cells increased from 0 to 2 CFU/mL, the fluorescence signal increased significantly. The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification. The detection limit of this method for S. Typhimurium in the poultry samples was 2 CFU/mL without any enrichment.     

RAPID SCREENING FOR SALMONELLA IN FISHMEAL BY ENRICHMENT-IMMUNOASSAY

We previously described an enrichment-immunoassay utilizing a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Here we evaluated it for the rapid screening for Salmonella in fishmeal obtained from the national Animal and Plant Quarantine service in the People's Republic of China. In this method, the number of Salmonella present is first expanded by appropriate enrichment cultures, and the pathogens are then directly detected by the T6 immunoassay. In a total of 94 enrichment cultures of fishmeal, we obtained an overall concordance of 98% between the results obtained in parallel by this method and by conventional test method. The positive prediction by this method was 92% and the negative prediction was 100%. The turn around time for the new test was 27 h which is a significant improvement from the turn around time exceeding 96 h required for the conventional test method. This test proved to be compatible with the routine work flow in the practical setting of a quarantine laboratory.     

A CHEMILUMINESCENCE BIOSENSOR COUPLED WITH IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM

A chemiluminescence biosensor, using a fiber-optic-linked photometer and a data acquisition unit connected to a PC, was developed in conjunction with immunomagnetic separation for rapid detection of Salmonella Typhimurium. Magnetic microbeads coated with Anti-Salmonella antibodies and anti-Salmonella antibodies conjugated with horseradish peroxidase (HRP) were added to artificially-inoculated samples, and the immuno-reaction was completed in 60 min resulting in a sandwich complex. A magnetic field was applied to collect magnetic beads and the addition of luminol to HRP-conjugated antibodies resulted in a chemiluminescence reaction. The signal was collected through a fiber optic light guide, measured with a photometer, and recorded in the data acquisition unit. The minimum detection limit of the chemiluminescence biosensor for S. Typhimurium was 1.97 × 10³ CFU/mL and the range of the detectable signal was from 8.6 to 350 mV for cell numbers from 1.97 × 10³ to 1.97 × 10⁶ CFU/mL. Signal values for 10⁶ CFU/mL of S. Typhimurium were at least 97 and 394% higher than the corresponding values for S. enteritidis and four times the signal values for others including S. montevideo, S. california, S. heidlberg, and S. seftenberg, respectively. The biosensor response showed a significant difference (P < 0.05) between 10³ CFU/mL S. Typhimurium and 10⁶ CFU/mL of commonly-occurring bacteria in foods including Listeria monocytogenes, Pseudomonas aeruginosa, Citrobacter freundii, Campylobacter jejuni, Escherichia coli O157, and generic Escherichia coli. A regression equation, V = 0.0262 N ^5.7713, with R²= 0.9713 was obtained for the calibration curve over the detection range for S. Typhimurium. The whole procedure could be completed within 90 min.     

应用特异等位PCR快速鉴定灰霉病菌抗苯莱特与乙霉威菌株*

根据灰霉病菌Botrytis cinerea对苯并咪唑类（苯莱特）及N－苯氨基甲酸酯类（乙霉威）杀菌剂的抗性与其β－微管蛋白的198和200位密码子的单碱基突变紧密相关的原理,建立了一种同时检测灰霉病菌对这两类杀菌剂抗性表型的特异等位聚合酶链式反应（ASP,Allele-specific Polymerase Chain Reaction）方法,即将突变的单碱基作为正引物的3'末端,与之对应的未突变的单碱基作为负引物的3'末端, 自行设计了LP4～LP8等五个等位点特异性引物,应用包括这五个引物在内的七组引物,对生测为BenSNPCR、BenHRNPCS和BenHRNPCR菌株进行了ASP扩增,结果表明：该方法可以检测出相应抗药表型的菌株,并能有效地检测田间灰霉病菌的抗药性,从而该方法克服了生物检测方法费时和准确性差等缺点。该方法还能鉴定出生测法确定为同一表型如BenHRNPCR 中的BenSNPCR异核菌丝,或灰霉病菌侵染寄主后所产生的回复突变,因此其方法的灵敏度极高,可应用于灰霉病菌抗药性分子生态学机理研究和田间抗性菌株的快速鉴定。由于未发现BenMRNPCR型菌株,其适用性有待进一步验证。