Hee Sung Hwang

A two-step quantitative pathogen detection system based on capillary electrophoresis.

Rapid identification of bacterial pathogens is important for patient management and initiation of appropriate antibiotic therapy in the early stages of infection. Among the several techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis combined with small subunit rRNA gene-specific polymerase chain reaction (PCR) has come into the spotlight owing to its sensitivity, resolution, and reproducibility. Despite the advantages of the method, the design of PCR primers and optimization of multiplex PCR conditions remain to be studied so that as many pathogens as possible can be analyzed in a single run. Here we describe a novel two-step technique involving multiplex PCR pathogen detection by CE-SSCP analysis followed by singleplex PCR pathogen quantification by CE-SSCP. Specific PCR primers were designed for optimal separation of their products by CE-SSCP based on molecular weight. PCR conditions were then optimized for multiplex analysis of the targets. Subsequently, detected pathogens were quantified by PCR with specific primers. Eight clinically important strains were simultaneously identified under the optimized conditions. Each individual pathogen was then quantified at a level of sensitivity of tens of cells per milliliter. In conclusion, the two-step pathogen detection method based on CE-SSCP described here allows for sensitive detection of pathogens by multiplex PCR (first step) and quantification by specific PCR (second step). The results illustrate the potential of the method in clinical applications.

A novel pathogen detection system based on high‐resolution CE‐SSCP using a triblock copolymer matrix

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.

Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP

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 102. These results indicate that high‐resolution CE‐SSCP systems have considerable potential in the clinical diagnosis of bacteria‐induced diseases.

Recent developments in CE-based detection methods for food-borne pathogens

Rapid and sensitive detection of food‐borne pathogens is critical for food safety from the viewpoint of both the public health professionals and the food industry. Conventional method is, however, known to be labor‐intensive, time‐consuming, and expensive due to the separate cultivation and biochemical assay. Many relevant technologies, such as flow cytometry, MALDI‐MS, ESI‐MS, DNA microarray, and CE, have been intensively developed to date. Among them, CE is considered to be the most efficient and reproducible because of low sample loss and simple automation. CE‐based pathogen detection methods can be classified into three categories based on the separation targets: cell separation, nucleic‐acid‐based identification, and protein separation coupled with characterization. In this review, recent developments in each sphere of CE‐based technology are discussed. Additionally, the critical features of each approach and necessary future technical improvements are also reviewed.

A new single-step quantitative pathogen detection system: template-tagging followed by multiplex asymmetric PCR using common primers and CE-SSCP.

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.

Triblock copolymer matrix-based capillary electrophoretic microdevice for high-resolution multiplex pathogen detection

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE‐SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer as a sieving matrix for CE‐SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO‐PPO‐PEO copolymers, 255‐bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO‐PPO‐PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan™ gel. Due to enhanced dynamic coating and sieving ability, PEO‐PPO‐PEO copolymer displayed fourfold enhancement of resolving power in the CE‐SSCP to separate same‐sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high‐resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO‐PPO‐PEO triblock copolymer an excellent matrix in the CE‐SSCP analysis on the microdevice.

Precise H1N1 swine influenza detection using stuffer-free multiplex ligation-dependent probe amplification in conformation-sensitive capillary electrophoresis

The H1N1 influenza virus has spread worldwide to become pandemic. Here, we developed a new method to discriminate various types of influenza A, including H1N1, using stuffer-free multiplex ligation-dependent probe amplification based on a conformation-sensitive separation method, namely capillary electrophoresis-single-strand conformation polymorphism. Unlike conventional methods, our approach precisely detects five relevant gene markers permitting confirmation of infection.

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.

Multiplex and Quantitative Pathogen Detection with High-Resolution Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism

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.

Micellar ordered structure effects on high-resolution CE-SSCP using Pluronic triblock copolymer blends

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.