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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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in the number of genes known to exhibit disease-associated aberrant methylation, the need for accurate multiplex assays for quantifying DNA methylation has increased. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is one method that has been highlighted in this context. However, two limitations make the custom design of MS-MLPA assays impractical: the need for long probes containing stuffer sequences and a reliance on only one restriction enzyme. Here, we developed a variation of MS-MLPA that employs a simpler probe-design process. To overcome the above-mentioned limitations, we used stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously; thus, probes satisfying desired criteria were available for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma. Our results showed that the custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels.

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is a prominent bioseparation method based on the mobility diversity caused by sequence-induced conformational differences of single-stranded DNA. The use of Pluronic polymer matrix has opened up new opportunities for CE-SSCP, because it improved the resolution for various genetic analyses. However, there still exists a challenge in optimizing Pluronic-based CE-SSCP, because the physical properties of Pluronic solutions are sensitive to temperature, particularly near the gelation temperature, where the viscoelasticity of Pluronic F108 solutions sharply changes from that of a Newtonian fluid to a hydrogel upon heating. We have focused on a set of experiments to control the ambient temperature of the CE system with the aim of enhancing the reliability of the CE-SSCP analysis by using the Applied Biosystems ABI 3130xl genetic analyzer with Pluronic F108 solution matrix. The ambient temperature control allowed us to vary the inlet and outlet portion of the capillary column, while the temperature of the column was kept at 35°C. The resolution to separate 2 single-base-pair-differing DNA fragments was significantly enhanced by changing the temperature from 19 to 30°C. The viscoelastic properties of the F108 solution matrix upon heating were also investigated by ex situ rheological experiments with an effort to reveal how the development of gels in Pluronic solutions affects the resolution of CE-SSCP. We found that the column inlet and outlet temperatures of the capillary column have to be controlled to optimize the resolution in CE-SSCP by using the Pluronic matrix., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Pluronic F108 block copolymers have shown a great promise to achieve the desirable high resolution in the conformation-sensitive separation of ssDNA using CE-SSCP. However, fundamental understanding of the structures and properties of Pluronic matrix affecting the resolution is still limited. Unlike conventional gel-forming homopolymers, Pluronic F108 block copolymers are amphiphilic macromolecules consisting of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers, which are capable of forming a highly ordered micellar structure in aqueous solution. In this study, we have performed a series of experiments by blending different types of Pluronic polymers to control the formation of micelles and to study the correlation between separation and rheological characteristics of Pluronic gels affecting the resolution of CE-SSCP. Our experiments have been specifically designed to elucidate how the micellar structure affects the resolution of CE-SSCP upon altering the size uniformity and constituent homogeneity of the micelles. Our results suggest that uniformly sized micelle packing is the primary structural feature of Pluronic gel matrix for the high-resolution separation, while the size and constituent of the micelle themselves need to be considered as secondary factors., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Among the molecular diagnostic methods for bacteria-induced diseases, capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) combined with 16S rRNA gene-specific PCR has enormous potential because it can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S rRNA gene-specific markers into separate peaks. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this report, a protocol for the detection of 12 pathogenic bacteria is provided. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing the easy identification of the pathogens.

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE-based SSCP (CE-SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene-specific markers unless combined with additional modification steps. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high-resolution CE-SSCP system using a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31-1.56 pg and a dynamic range of approximately 10(2). These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.

Although CE-SSCP analysis combined with 16S ribosomal RNA gene-specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE-SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE-SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE-SSCP analysis. A poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE-SSCP analysis using the PEO-PPO-PEO triblock copolymer as the polymer matrix and four same-sized DNA fragments of similar sequence content. Among 48 commercially available PEO-PPO-PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO(137)PPO(43)PEO(137) exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE-SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO-PPO-PEO triblock copolymer may serve as an ideal matrix for high-resolution CE-SSCP analysis.

Quantification of RNA provides information crucial for various biological studies, including analysis of mRNA expression and that of microRNAs. Reverse transcription (RT) coupled with real-time polymerase chain reaction (PCR) is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Here, we report a novel multiplex RNA quantification method that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) coupled with modified RT and asymmetric PCR. The reverse transcripts of seven in vitro transcribed RNAs were modified with common sequence tags and amplified by asymmetric PCR using primers specific to the common tags. The resulting amplicons were separated and quantified by CE-SSCP. A series of experiments using different amounts of RNA demonstrated that the assay had a limit of detection of 2 amol and a dynamic range of approximately 10(5). These results clearly indicate the potential of this method to provide robust and precise multiplex RNA quantification.

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria-induced disease. Among the existing techniques for identifying microbial, CE-SSCP combined with 16S ribosomal RNA gene-specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE-SSCP can separate PCR products with high-resolution, multiplex detection and quantification are complicated by primer-dimer formation and non-specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template-tagging followed by multiplex asymmetric PCR and subsequent CE-SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia-inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE-SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.

Conventional antimicrobial activity analyses such as the broth dilution method and disk diffusion test are considerably demanding processes for new antimicrobial agent discovery and sensitive diagnosis of infectious diseases. Here, we developed a new antimicrobial activity analysis system using CE-based SSCP (CE-SSCP) combined with 16S rRNA gene-specific PCR (PCR/CE-SSCP). Using this method, the population change in the microbial community in response to specific antimicrobial agents could be quantified with a high sensitivity and accuracy from a small sample amount. Using a mixture of microorganisms comprising Escherichia coli, Corynebacterium glutamicum, Acinetobacter calcoaceticus, and Staphylococcus aureus as a model system, the linear correlation between the genomic DNA concentrations and peak areas in 16S rRNA gene-specific PCR/CE-SSCP was determined; consequently, quantification of cell concentrations could be demonstrated using this method. Compared to the minimum inhibitory concentration (MIC) values from the conventional broth dilution method, this new system provided almost the same MIC values for popular antimicrobial agents such as kanamycin, spectinomycin, and streptomycin. The results demonstrated that the newly developed method can be a substitute for the conventional antimicrobial analysis method and highlighted its high potential in the areas of new antimicrobial agent discovery and clinical diagnosis.