Compact, reliable asymmetric optical configuration for cost-effective fabrication of multiplex dot matrix hologram in anti-counterfeiting applications

This study presents a compact optical configuration that can generate a multiplex dot-matrix hologram with complex interlaced images for anti-counterfeiting applications on valuable paper. Varying the orientation of the interference plane can enable the multiplex hologram to be recorded without changing the interference angle of two laser beams. With its simple asymmetric optical setup, a multiplex hologram with many interlaced images can be efficiently fabricated, increasing the cost of imitation. Experimental details of the asymmetric optical setup are also described.     

Automatic analysis of multiplex ligation-dependent probe amplification products (exemplified by a commercial kit for prenatal aneuploidy detection)

For use in routine prenatal diagnostics, we developed software and methods for automatic aneuploidy detection based on a commercial multiplex ligation-dependent probe amplification (MLPA) kit. Software and methods ensure a reliable, objective, and fast workflow, and may be applied to other types of MLPA kits. Following CE of MLPA amplification products, the software automatically identified the peak area for each probe, normalized it in relation to the neighboring peak areas of the test sample, computed the ratio relative to a reference created from normal samples, and compensated the ratio for a side effect of the normalization procedure that scaled all chromosomally normal DNA peak areas slightly up or down depending on the kind of aneuploidy present. For the chromosomes 13, 18, 21, X, and Y, probe reliability weighted mean ratio values and corresponding SDs were calculated, and the significance for being outside a reference interval around ratio 1.0 was tested. p < or = 1% suggested aneuploidy and 1 < p < or = 5% suggested potential aneuploidy. Individual peaks, where the normalized area was situated more than 4 SD from the corresponding reference, suggested possible partial deletion or gain. Sample quality was automatically assessed. Control probes were not required. Having used the software and methods for two years, we conclude that a reliable, objective, and fast workflow is obtained.     

The development of an 18-locus Y-STR system for forensic casework

The aim of the present work was to improve the discriminatory potential, and hence the probative value, of Y-STR-based testing by extending the set of Y chromosome STR loci available for forensic casework. In accordance with the requirements of a Y chromosome multiplex analytical system developed specifically for forensic casework use, we have sought to maximize the number of loci able to be co-amplified, ensure appropriate assay sensitivity (1–2 ng of input genomic DNA), balance inter-locus signals and minimize confounding female DNA artifacts. Two Y chromosome STR systems, multiplex I (MPI) and multiplex II (MPII), have been developed which permit the robust co-amplification of 18 Y-STRs. The loci include DYS19, DYS385(a) and (b), DYS388, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS425, DYS434, DYS437, DYS438, DYS439, Y-GATA-C4, Y-GATA-A7.1 (DYS460) and Y-GATA-H4. The two multiplex systems are robust over a wide range of primer, magnesium, and DNA polymerase concentrations and perform well under a variety of cycling conditions. Complete male haplotypes can be obtained with as little as 100–250 pg of template DNA. Although a limited number of female DNA artifacts are observed in mixed stains in which the male DNA comprises 1/100 of the total, the male profile is easily discernible. Slightly modified versions of MPI and MPII demonstrate a significant reduction in female artifacts. Thus, it may not be necessary to employ a differential extraction strategy to obtain a male haplotype (or haplotypes in the case of multiple male donors) in cases of sexual assault. The potential utility of MPI and MPII for forensic casework is exemplified by their ability to dissect out the male haplotype in post-coital vaginal swabs and to determine the number of male donors in mixed semen stains.This study has emphasized the need for novel Y-STR multiplexes developed for forensic use to undergo a series of validation exercises that go beyond simply optimizing the PCR reaction conditions. Specifically, stringent performance checks on their efficacy need to be carried out using casework-type specimens in order to determine potential confounding effects from female DNA.     

Automated,high performance,flow-through chemiluminescence microarray for the multiplexed detection of phycotoxins

A novel multiplexed immunoassay for the analysis of phycotoxins in shellfish samples has been developed. Therefore, a regenerable chemiluminescence (CL) microarray was established which is able to analyze automatically three different phycotoxins (domoic acid (DA), okadaic acid (OA) and saxitoxin (STX)) in parallel on the analysis platform MCR3. As a test format an indirect competitive immunoassay format was applied. These phycotoxins were directly immobilized on an epoxy-activated PEG chip surface. The parallel analysis was enabled by the simultaneous addition of all analytes and specific antibodies on one microarray chip. After the competitive reaction, the CL signal was recorded by a CCD camera. Due to the ability to regenerate the toxin microarray, internal calibrations of phycotoxins in parallel were performed using the same microarray chip, which was suitable for 25 consecutive measurements. For the three target phycotoxins multi-analyte calibration curves were generated. In extracted shellfish matrix, the determined LODs for DA, OA and STX with values of 0.5 ± 0.3 μg L⁻¹, 1.0 ± 0.6 μg L⁻¹, and 0.4 ± 0.2 μg L⁻¹ were slightly lower than in PBS buffer. For determination of toxin recoveries, the observed signal loss in the regeneration was corrected. After applying mathematical corrections spiked shellfish samples were quantified with recoveries for DA, OA, and STX of 86.2%, 102.5%, and 61.6%, respectively, in 20 min. This is the first demonstration of an antibody based phycotoxin microarray.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Rapid and sensitive detection of lower respiratory tract infections by stuffer‐free multiplex ligation‐dependent probe amplification

Lower respiratory tract infection is one of the most common infectious diseases. However, conventional methods for detecting infectious pathogens are time‐consuming, and generally have a limited impact on early therapeutic decisions. We previously reported a rapid and sensitive method for detecting such pathogens using stuffer‐free multiplex ligation‐dependent probe amplification coupled with high‐resolution CE‐SSCP. In this study, we report an application of this method to the detection of respiratory pathogens. As originally configured, this method was capable of simultaneously detecting seven bacterial species responsible for lower respiratory tract infections, but its detection limit and assay time were insufficient to provide useful information for early therapeutic decisions. To improve sensitivity and shorten assay time, we added a target‐specific preamplification step, improving the detection limit from 50 pg of genomic DNA to 500 fg. We further decreased time requirements by optimizing the hybridization step, enabling the entire assay to be completed within 7 h while maintaining the same detection limit. Taken together, these improvements enable the rapid detection of infectious doses of pathogens (i.e. a few dozen cells), establishing the strong potential of the refined method, particularly for aiding early treatment decisions.     

NMR analysis of multiple samples using parallel coils: improved performance using reference deconvolution and multidimensional methods

Improvements in the Multiplex Sample NMR method are investigated to explore its capabilities of analyzing multiple samples simultaneously. Issues of quantitation and resolution in the multiple-coil probe are examined in one- and two-dimensional experiments. Improvements in quantitation are shown to result from the use of reference deconvolution for one-dimensional experiments, while the use of two-dimensional methods has much improved resolution and shows the potential for significantly increased parallelism. A multiplicative scheme is shown to be an easily implemented, effective method for generating individual sub-spectra from individual samples.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

Multiplex bioanalytical methods for food and environmental monitoring

Recent advances in miniaturization of analytical systems and newly emerging technologies offer platforms with greater automation and multiplexing capabilities than traditional biological binding assays. Multiplexed bioanalytical techniques provide control agencies and food industries with new possibilities for improved, more efficient monitoring of food and environmental contaminants. This review deals with recent developments in planar-array and suspension-array technologies, and their applications in detecting pathogens, food allergens and adulterants, toxins, antibiotics and environmental contaminants.