Amplification of microsphere-based microarrays using catalyzed reporter deposition

Assay sensitivities using three fluorescent signal generation schemes were evaluated on the Luminex flow cytometer. Following microsphere capture of antigen by immobilized antibodies, bound targets were quantified by use of (1) Cy3-labeled "tracer" antibodies (30min total time), (2) biotinylated tracers followed by streptavidin-R-phycoerythrin (60min total time), or (3) biotinylated tracers followed by avidin-peroxidase conjugates and tyramide signal amplification (TSA; 90min total time). Use of TSA for signal generation in three individual toxin assays improved performance up to 100-fold over Cy3-antibody-based detection, and while streptavidin-R-phycoerythrin provided equivalent sensitivities, TSA produced dramatic increases at low concentrations simplifying positive sample identification. Detection limits for TSA-interrogated assays for ricin, cholera toxin, and staphylococcal enterotoxin B were 64pg/ml, 4pg/ml, and 0.1ng/ml, respectively, using optimized conjugates; analogous detection limits for Cy3-antibody-interrogated assays were 8ng/ml, 1ng/ml, and 1ng/ml, respectively. No improvement was observed in botulinum toxoid A assays when TSA amplification was used. As unique preferences for specific avidin-peroxidase conjugates were observed in the individual assays, improvements in multiplexed assays utilizing a single conjugate were significantly lower (3-10-fold improvements). Furthermore, increases in variability resulted in poorer performance of TSA-interrogated assays for botulinum toxoid, indicating that assay-specific optimization should be performed, especially prior to multiplexing.     

Biotechnology offers revolution to fish health management

Biotechnology has many applications in fish health management. The application of monoclonal antibodies (mAbs) provides a rapid means of pathogen identification; antibodies to immunoglobulins from different fish species can be used to monitor the host response following vaccination; and mAbs also have the potential for screening broodstock for previous exposure to pathogens. Luminex technology exemplifies a novel antibody-based method that can be applied to both pathogen detection and vaccine development. Molecular technologies, such as the polymerase chain reaction (PCR), real time PCR and nucleic acid sequence-based amplification (NASBA), have enabled detection, identification and quantification of extremely low levels of aquatic pathogens, and microarray technologies offer a new dimension to multiplex screening for pathogens and host response. Recombinant DNA technology permits large-scale, low-cost vaccine production, moreover DNA vaccination, proteomics, adjuvant design and oral vaccine delivery will undoubtedly foster the development of effective fish vaccines in the future.     

New methods for the diagnosis of enteric infections

Gastrointestinal disease remains a major cause of mortality and morbidity throughout the world. Recently, a number of viral, bacterial, and protozoan agents have been identified which can cause a range of gastrointestinal disorders. The effective management of these diseases requires the prompt identification of the infecting micro-organism and the early institution of preventative and therapeutic interventions. The detection of infecting microorganisms in fecal and intestinal fluids presents a particular challenge to the diagnostic microbiologist. Cultivation can be difficult due to the fastidious nature of the microorganisms and the presence of cytotoxic materials in the specimen. In the past, immunoassays have been used for the detection of some microorganisms. However, immunoassays have limited sensitivity and cannot detect all infecting microorganisms. Recently, nucleic acid amplification techniques have been developed for the direct detection of pathogenic microbial DNA and RNA in human body fluids. We have found that these methods can be applied for the accurate detection of intestinal vlruses provided that inhibitors of enzymatic amplification are removed from the sample. Using affinity binding purification and non-isotopic DNA measurement techniques, we have developed sensitive and specific assays for the quantitation of a wide range of infecting microorganisms in intestinal fluids. Nucleic acid amplification provides a Abstract continued on next page unique tool for the study of enteric pathogens and for the development of strategies for their eventual elimination from the human environment.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.     

Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.

BACKGROUND: Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation. METHODS: The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead. RESULTS: The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated. CONCLUSIONS: Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Detecting antigens by quantitative immuno-PCR

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.     

Dry reagent dipstick test combined with 23S rRNA PCR for molecular diagnosis of bacterial infection in arthroplasty

Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chain reaction (PCR), followed by electrophoretic analysis of the products, have been introduced. We report a simple and inexpensive assay that allows visual detection and confirmation of the PCR-amplified sequences by hybridization within minutes. The assay is performed in a dry reagent dipstick format (strip) and does not require special instrumentation. Universal primers are used for PCR of the 23S ribosomal RNA (rRNA) gene. The biotinylated amplification product is hybridized with dA-tailed probes that are specific for six pathogens commonly involved in periprosthetic joint infections. The mixture is applied to the strip, which is then immersed in the appropriate buffer. The buffer migrates along the strip by capillary action and rehydrates gold nanoparticles with oligo(dT) strands attached to their surface. The nanoparticles bind to the target DNA through hybridization, and the hybrids are captured by immobilized streptavidin at the test zone of the strip, producing a characteristic red line. Unbound nanoparticles are captured by immobilized oligo(dT) strands at the control zone of the strip, generating a second line. The dipstick test was applied to the detection of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faesium, and Haemophilus influenza. Twelve samples of synovial fluids from patients were analyzed for the detection and identification of the infection caused by the six pathogens. The results were compared with bacterial cultures.     

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.     

Molecular detection and phylogenetic placement of a microsporidian from English sole (Pleuronectes vetulus) affected by X-cell pseudotumors

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.     

Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.     

环介导等温基因扩增技术及其在病毒检测中的应用

环介导等温扩增是一门新兴的分子生物学检测技术,因其具有特异性高、敏感性高、简单、快捷及不需要昂贵的仪器设备等特点,受到了生物医学研究者的高度关注。目前,该方法已经被广泛应用于各种病原微生物检测。简要综述了环介导等温扩增技术的原理、特点,及其在病毒检测中的应用。     

DNA probes for the identification of microorganisms

Summary The detection and identification of microorganisms is being carried out increasingly using DNA. Each organism has a unique DNA sequence which can be used to distinguish closely related organisms. Using PCR amplification and sequencing of ribosomal RNA genes we have developed DNA probes for a number of pathogenic bacteria and fungi. The development of DNA assays based on PCR has resulted in new questions which must be addressed including process carry-over contamination and inhibition of the PCR amplification reaction once the problems associated with the implementation of DNA assays are ironed out.     

环介导等温扩增核酸技术及其应用

环介导等温扩增(loop-mediated isothermal amplification,简称LAMP)是利用4个特殊设计的引物和具有链置换活性的DNA聚合酶,在恒温条件下特异、高效、快速地扩增DNA的新技术。该技术在1h内能扩增出109靶序列拷贝,扩增产物是一系列反向重复的靶序列构成的茎环结构和多环花椰菜样结构的DNA片段的混合物,电泳后在凝胶上显现出由不同大小的区带组成的阶梯式图谱。LAMP技术以其特异性强、灵敏度高、快速、准确和操作简便等优点在核酸的科学研究、疾病的诊断和转基因食品检测等领域得到了日益广泛的应用。     

DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT + Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays.     

滚环扩增信号放大技术在生物检测中应用的研究进展

滚环扩增(Rolling circle amplification,RCA)是一种快速、灵敏且恒温的单链DNA(Single-stranded DNA,ssDNA)扩增技术,与染色或探针联用可实现检测信号的放大,在生物检测等方面得到广泛的应用。文中对RCA的构建方法进行了简介,综述了近几年其在致病菌、核酸肿瘤标记物、蛋白质、生物小分子和病毒等检测中的研究进展,并对其未来的发展趋势进行了展望。     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.     

Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 × 10⁶ genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.     

Bacterial Type IV secretion systems: versatile virulence machines

Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile Type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated 'effectors' vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.     

Research progress in the detection of common foodborne hazardous substances based on functional nucleic acids biosensors

With the further improvement of food safety requirements, the development of fast, highly sensitive, and portable methods for the determination of foodborne hazardous substances has become a new trend in the food industry. In recent years, biosensors and platforms based on functional nucleic acids, along with a range of signal amplification devices and methods, have been established to enable rapid and sensitive determination of specific substances in samples, opening up a new avenue of analysis and detection. In this paper, functional nucleic acid types including aptamers, deoxyribozymes, and G-quadruplexes which are commonly used in the detection of food source pollutants are introduced. Signal amplification elements include quantum dots, noble metal nanoparticles, magnetic nanoparticles, DNA walkers, and DNA logic gates. Signal amplification technologies including nucleic acid isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, biological barcodes, and microfluidic system are combined with functional nucleic acids sensors and applied to the detection of many foodborne hazardous substances, such as foodborne pathogens, mycotoxins, residual antibiotics, residual pesticides, industrial pollutants, heavy metals, and allergens. Finally, the potential opportunities and broad prospects of functional nucleic acids biosensors in the field of food analysis are discussed.     

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.