New techniques in rapid viral diagnosis

Abstract The development of new diagnostic techniques in immunology and molecular biology during the last two decades has opened up new possibilities for rapid viral diagnosis. Solid phase immunoassays for antigen and antibody detection are now widely used in diagnostic settings. Several novel techniques have been introduced and have led to commercially available tests. Diagnostic methods using nucleic acid amplification procedures are already applied in research laboratories and will be commercialized soon. Biosensor-based diagnostic techniques have the potential of generating a result nearly instantaneously and it has become possible to monitor kinetic processes. Automatization and simplified procedures are needed to allow diagnostic tests to be performed soon after the sample has been obtained from the patient. In order to evaluate the new procedures and avoid false results, rigorous quality control in diagnostic virology will have to be instituted.     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

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.     

What's new: Ligation-based DNA diagnostics

A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of probe molecules. The probes are designed to hybridize immediately next to each other on the target nucleic acid strand. Demonstration of ligated probes results in highly specific detection of and efficient distinction between similar sequence variants under standard reaction conditions. Accordingly, the principle has been applied in automated genetic screening procedures. Ligation reactions are also integral to a number of amplification procedures and they will be of value in an expanding range of genetic analyses.     

Amplification methods to increase the sensitivity of in situ hybridization: play card(s).

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes

Nucleic acid diagnostics is dominated by fluorescence-based assays that use complex and expensive enzyme-based target or signal-amplification procedures. Many clinical diagnostic applications will require simpler, inexpensive assays that can be done in a screening mode. We have developed a 'spot-and-read' colorimetric detection method for identifying nucleic acid sequences based on the distance-dependent optical properties of gold nanoparticles. In this assay, nucleic acid targets are recognized by DNA-modified gold probes, which undergo a color change that is visually detectable when the solutions are spotted onto an illuminated glass waveguide. This scatter-based method enables detection of zeptomole quantities of nucleic acid targets without target or signal amplification when coupled to an improved hybridization method that facilitates probe-target binding in a homogeneous format. In comparison to a previously reported absorbance-based method, this method increases detection sensitivity by over four orders of magnitude. We have applied this method to the rapid detection of mecA in methicillin-resistant Staphylococcus aureus genomic DNA samples.     

Real-time monitoring of the strand displacement amplification (SDA) of human cytomegalovirus by a new SDA-piezoelectric DNA sensor system

Nucleic acid amplification has long been used in biosensor technologies, such as DNA sensors, DNA chips and microarrays, due to its advantage of high sensitivity in detecting target DNA. However, dynamic monitoring of nucleic acid amplifications with traditional DNA sensors in real-time is difficult since a constant temperature must be maintained during detection. Thus, the piezoelectric sensor, one type of traditional DNA sensor, is not applicable in real-time monitoring PCR due to the dramatic change in temperature that occurs during reaction. In this study, we introduced strand displacement amplification (SDA), an well-developed nucleic acid amplification technique that can work under conditions of constant temperature, into the development of a novel piezoelectric sensor. Using the new SDA-piezoelectric DNA sensor, we designed a stable system for liquid-phase detection, in which the crystal oscillator plate was fixed by an easily adjustable screw-threaded clamping mechanism and successfully applied the new sensor system to real-time SDA monitoring of human cytomegalovirus (HCMV). This new technique overcomes the shortcomings of traditional DNA sensors in real-time monitoring of nucleic acid amplification. The technique has proved to be a markedly simplified procedure with a number of advantages, such as higher sensitivity, better time efficiency, and the ability of dynamic real-time detection.     

DNA detection using recombination proteins

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.     

Signal amplification by rolling circle amplification on DNA microarrays

While microarrays hold considerable promise in large-scale biology on account of their massively parallel analytical nature, there is a need for compatible signal amplification procedures to increase sensitivity without loss of multiplexing. Rolling circle amplification (RCA) is a molecular amplification method with the unique property of product localization. This report describes the application of RCA signal amplification for multiplexed, direct detection and quantitation of nucleic acid targets on planar glass and gel-coated microarrays. As few as 150 molecules bound to the surface of microarrays can be detected using RCA. Because of the linear kinetics of RCA, nucleic acid target molecules may be measured with a dynamic range of four orders of magnitude. Consequently, RCA is a promising technology for the direct measurement of nucleic acids on microarrays without the need for a potentially biasing preamplification step.     

Feasibility of methods based on nucleic acid amplification techniques to fulfil the requirements for microbiological analysis of water quality

Molecular methods based on nucleic acid recognition and amplification are valuable tools to complement and support water management decisions. At present, these decisions are mostly supported by the principle of end‐point monitoring for indicators and a small number of selected measured by traditional methods. Nucleic acid methods show enormous potential for identifying isolates from conventional culture methods, providing data on cultivable and noncultivable micro‐organisms, informing on the presence of pathogens in waters, determining the causes of waterborne outbreaks, and, in some cases, detecting emerging pathogens. However, some features of water microbiology affect the performance of nucleic acid‐based molecular techniques and thus challenge their suitability for routine water quality control. These features include the variable composition of target water samples, the generally low numbers of target micro‐organisms, the variable water quality required for different uses and the physiological status or condition of such micro‐organisms. The standardization of these molecular techniques is also an important challenge for its routine use in terms of accuracy (trueness and precision) and robustness (reproducibility and reliability during normal usage). Most of national and international water regulations recommend the application of standard methods, and any new technique must be validated respect to established methods and procedures. Moreover, molecular methods show a high cost‐effectiveness value that limits its practicability on some microbial water analyses. However, new molecular techniques could contribute with new information or at least to supplement the limitation of traditional culture‐based methods. Undoubtedly, challenges for these nucleic acid‐based methods need to be identified and solved to improve their feasibility for routine microbial water monitoring.     

Study of the structural-energy aspects of complementary-addressed modifications of nucleic acids by reactive oligonucleotide derivatives having a 4-[N-methyl-N-(2-chloroethyl)amino[benzylidene group at the 3'end by a molecular mechanics method

Calculations of probabilities of the complementary addressed modification of a target nucleic acid by derivatives of oligonucleotides carrying a 4-[N-(2-chloroethyl)-N-methyl]aminobenzylidene group attached to the 3'-end (3'-BDO) have been made. The results show that the complementary complex of a target NA with 3'-BDO having R-configuration of carbon atom of the dioxalane ring is more stable than the complex including the S-stereoisomer. The S- and R-epimeres of 3'-BDO have essentially different positional abilities for alkylation of the target. The R-epimer alkylates best of all the third base of the target NA from terminal complementary pair of the complex. The S-epimer has another site the most sensitive to alkylation, which is a terminal complementary base of the target NA or the adjacent nonpaired base. Formation of the alkylation complexes are accompanied with a loss or a breakdown of hydrogen bonds in the terminal complementary base pair, thus decreasing the efficiency of alkylation. The modelling results are considered along with experimental data on modification which therefore can be interpreted on the fundamental structural level.     

Strand Invasion Based Amplification (SIBA?): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.     

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization

Background

State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.

Methodology and Principal Findings

The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.

Conclusions and Significance

The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.