Quantitative detection of residual E. coli host cell DNA by real-time PCR

E. coli has been widely used as a host system to manufacture recombinant proteins for human therapeutic use. Among impurities to be eliminated during the downstream process, residual host cell DNA is a major interest for safety. Residual E. coli host cell DNA in the final products are usually determined using conventional slot blot hybridization assay or total DNA Threshold assay, although these methods are time consuming, expensive, and relatively insensitive. Therefore a sensitive real-time PCR assay for specific detection of residual E. coli DNA was developed and compared with slot blot hybridization assay and Threshold assay to validate the overall capability of these methods. Specific primer pair for amplification of the E. coli 16S rRNA gene was selected to improve the sensitivity, and E. coli host cell DNA was quantified by use of SYBR Green 1. The detection limit of real-time PCR assay in the optimized condition was calculated to be 0.042 pg genomic DNA, which is much higher than those of slot blot hybridization assay and Threshold assay of which detection limit were 2.42 and 3.73 pg genomic DNA, respectively. The real-time PCR assay was validated to be more reproducible, accurate, and precise than slot blot hybridization assay and Threshold assay. The real-time PCR assay may be a useful tool for quantitative detection and clearance validation of residual E. coli DNA during the manufacturing process for recombinant therapeutics.     

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Detection of microarray-hybridized oligonucleotides with magnetic beads

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.     

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.     

A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.     

Highly sensitive and specific detection of viable Escherichia coli in drinking water

A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.     

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH(2)) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β-d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0×10(-10) mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×10(2) to 2.0×10(3) cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.     

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay.     

Stationary phase protein overproduction is a fundamental capability of Escherichia coli

Although Escherichia coli is well studied and various recombinant E. coli protein expression systems have been developed, people usually consider the rapid growing (log phase) culture of E. coli as optimum for production of proteins. However, here we demonstrate that at stationary phase three E. coli systems, BL21 (DE3)(pET), DH5alpha (pGEX) induced with lactose, and TG1 (pBV220) induced with heat shock could overexpress diversified genes, including three whose products are deleterious to the host cells, more stably and profitably than following the log phase induction protocol. Physical and patch-clamp assays indicated that characteristics of target proteins prepared from cultures of the two different growth phases coincide. These results not only provide a better strategy for recombinant protein preparation in E. coli, but also reveal that rapid rehabilitation from stresses and stationary phase protein overproduction are fundamental characters of E. coli.     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列[醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes.

A dot-blot hybridization immunoenzymatic assay with a chemiluminescent endpoint was developed for the rapid and sensitive detection of viral and plasmid DNAs. Digoxigenin-labeled probes were used to detect cytomegalovirus, parvovirus B19, and plasmid pBR328 DNAs. Hybridized probes were immunoenzymatically visualized by anti-digoxigenin Fab fragments labeled with alkaline phosphatase, and adamantyl 1,2-dioxetane phenyl phosphate was used as chemiluminescent substrate. Results were recorded by instant photographic films. The chemiluminescent hybridization assay was performed in about 8 hr and was able to detect as little as 50-10 fg of homologous target DNA.     

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.

A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.     

Specific detection of Escherichia coli and Shigella species using fragments of genes coding for β-glucuronidase

Abstract The occurrence of β-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli , coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and ampplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes fro E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei ), independent of the β-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 × 10⁴ bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.     

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Detection of Escherichia coli in sewage and sludge by polymerase chain reaction.

A method in which the polymerase chain reaction (PCR) was used was developed to amplify either a uidA gene fragment or a 16S rRNA gene fragment from Escherichia coli in sewage and sludge. Because of interference caused by humic acidlike substances, crude DNA extracts were purified with a Sephadex G-200 spun column before the PCR was begun. A Southern analysis in which a nonradioactive chemiluminescent method was used was performed to confirm the presence of PCR products. The sensitivity of detection for PCR products when the chemiluminescent method was used was determined to be 30 ag of E. coli genomic DNA template. In seeded sludge, the PCR amplified the target DNA from 80 E. coli cells per g of sludge and 50 Shigella dysenteriae cells per g of sludge. Because only 0.05 aliquot of a sludge extract was used for the PCR, we deduced that the PCR detected target DNA equivalent to the DNA of 2.5 to 4 cells in the extract. The PCR amplified the uidA fragment from diluted sewage influents and effluents containing E. coli cells. Therefore, the PCR performed with a chemiluminescent gene probe can be used to detect the presence of potentially pathogenic microorganisms in sewage and sludge. This technique can be expanded to permit direct detection of pathogenic microorganisms in water samples, thus leading to enhanced public health protection.     

Quantification of Escherichia coli genomic DNA contamination in recombinant protein preparations by polymerase chain reaction and affinity-based collection

This study describes the development of a novel assay for the quantification of Escherichia coli genomic DNA contamination in recombinant protein samples. The technique is based on PCR amplification and digoxygenin labeling of the genes encoding 5S ribosomal RNA followed by affinity-based collection and detection. Samples containing 1 pg x mL(-1) of extracted E. coli genomic DNA (gDNA) could be measured using this method. Using extracted E. coli gDNA as standards, a 35-cycle PCR reaction exhibited a linear response versus template concentration between 1 pg x mL(-1) and1 ng x mL(-1) genomic DNA even when diluted in a variety of buffering conditions. Comparison of the novel assay with a traditional filter binding and hybridization technique using recombinant protein samples confirmed that the procedure was accurate and sensitive. The assay described in this report is a safer and less expensive alternative to radioactive techniques employed for DNA quantification, utilizing readily available reagents and apparatus.