Stuffer‐free multiplex ligation‐dependent probe amplification based on conformation‐sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation

Developing diagnostic tools based on the application of known disease/phenotype‐associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user‐friendly multiplex CNV detection method, termed stuffer‐free MLPA‐CE‐SSCP, that combines a variation of multiplex ligation‐dependent probe amplification (MLPA) with CE‐SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar‐sized stuffer‐free MLPA products, we adopted CE‐SSCP rather than length‐dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X‐chromosome copy number (1–5) revealed that copy number determinations using stuffer‐free MLPA‐CE‐SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.     

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.     

Multiplex identification of drug‐resistant Gram‐positive pathogens using stuffer‐free MLPA system

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug‐resistant Gram‐positive pathogens, we developed a method by using stuffer‐free multiplex ligation‐dependent probe amplification (MLPA) coupled with high‐resolution CE single‐strand conformation polymorphisms (CE‐SSCP) system. We designed three probe sets for genes specific to Gram‐positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram‐positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain‐specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target‐specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug‐resistant Gram‐positive pathogens from sepsis patients’ blood.     

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.     

Development of solution‐dispersible hyperbranched conjugated polymer nanoparticles for Fe3+ fluorescent detection and their application in logic gate

Solution‐dispersible hyperbranched conjugated polymer nanoparticles (FT‐HBCPNs) consist of an intrinsic crosslinked rigid skeleton structure of both 9,9‐dihexyl‐fluorene and triphenylamine repeating units, and are synthesized via the miniemulsion Suzuki polymerization, and FT‐HBCPNs for highly selective and sensitive Fe³⁺ fluorescent detection and their application in logic gate at molecular level are successfully developed. FT‐HBCPNs with an average particle size of 10.6 nm can disperse in common organic solvents. FT‐HBCPNs show high selectivity and sensitivity for Fe³⁺ over other commonly co‐existent metal ions in THF solution with a detection limit of 3.65 × 10^?8 mol L^?1. Furthermore, homogeneous transparent thin films of FT‐HBCPNs developed by a simple spin‐coating method can be reversibly quenched by Fe³⁺ with a detection limit of 3.09 × 10^?7 mol L^?1. Using Fe³⁺ and EDTA as chemical inputs and the fluorescence intensity signal as outputs, FT‐HBCPNs films can be utilized as a logic gate at molecular level. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3694–3700     

Synthesis and characterization of polyvinylpyrrolidone immobilized on magnetic nanoparticles modified by ionic liquid as a novel and recyclable catalyst for the three‐component synthesis of amidoalkyl naphthols

In this study, an efficient and green procedure is explained for the preparation of 1‐amidoalkyl‐2‐naphthols applying one‐pot condensation reaction of 2‐naphthol, amide and aromatic nanoparticles (Fe₃O₄@SiO₂@IL‐PVP) as a novel solid acid catalyst under solvent‐free conditions. The remarkable features of this method are short reaction time, high conversions, and high yield of product, easy workup procedures and solvent‐free conditions. The Fe₃O₄@SiO₂@IL‐PVP catalyst was characterized via Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffraction patterns (XRD), scanning electron microscopy (SEM), Transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), vibrating sample magnetometer (VSM), and energy‐dispersive X‐ray spectroscopy (EDS). Also, nanocatalysts could be easily recovered by a simple magnet and reused for the next reactions without significant loss of its catalytic activity.     

Development of a ligase detection reaction/CGE method using a LIF dual‐channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild‐type DNA

We developed an inexpensive LIF dual‐channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low‐abundance point mutations in a large excess of wild‐type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K‐ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K‐ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild‐type sequence‐dominated population, and to disclose their allelic composition, was thus demonstrated successfully.