Boram Chung

Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay.

We developed a novel 3D immunomagnetic flow assay for the rapid detection of pathogenic bacteria in a large-volume food sample. Antibody-functionalized magnetic nanoparticle clusters (AbMNCs) were magnetically immobilized on the surfaces of a 3D-printed cylindrical microchannel. The injection of a Salmonella-spiked sample solution into the microchannel produced instant binding between the AbMNCs and the Salmonella bacteria due to their efficient collisions. Nearly perfect capture of the AbMNCs and AbMNCs-Salmonella complexes was achieved under a high flow rate by stacking permanent magnets with spacers inside the cylindrical separator to maximize the magnetic force. The concentration of the bacteria in solution was determined using ATP luminescence measurements. The detection limit was better than 10 cfu/mL, and the overall assay time, including the binding, rinsing, and detection steps for a 10 mL sample took less than 3 min. To our knowledge, the 3D immunomagnetic flow assay described here provides the fastest high-sensitivity, high-capacity method for the detection of pathogenic bacteria.

Multiplex quantitative foodborne pathogen detection using high resolution CE-SSCP coupled stuffer-free multiplex ligation-dependent probe amplification.

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.

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.

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.

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.

Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Antibody‐conjugated magnetic nanoparticles (Ab‐MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab‐MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab‐MNPs as a pathogen detection method requires accurate characterization of Ab‐MNP capture ability and standardization of all handling processes. In this study, we used high‐resolution CE‐single strand conformational polymorphism coupled with a stuffer‐free multiplex ligation‐dependent probe amplification system to characterize Ab‐MNPs. The capture ability of Ab‐MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab‐MNPs was also assessed. The results showed that the stuffer‐free multiplex ligation‐dependent probe amplification system has the potential to serve as a standard characterization method for Ab‐MNPs. Moreover, the precise characterization of Ab‐MNPs facilitated robust pathogen detection in various applications.

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.

Triblock copolymer‐based microchip device for rapid analysis of stuffer‐free multiplex ligation‐dependent probe amplification products

Recent improvements in the multiplex ligation‐dependent probe amplification (MLPA) method promise successful multiplex analysis of various genetic markers. In particular, it has been demonstrated that elimination of the stuffer sequence included in MLPA probes for length‐dependent analysis substantially simplifies the probe design process and improves the accuracy of the analysis. As is the case for other CE‐based methods, MLPA could be further developed on a microchip platform. However, high‐resolution analysis of short MLPA probes requires careful microchip operation. In this study, we developed a microchip device for the multiplex analysis of five food‐borne pathogens using a stuffer‐free probe set. Microchip channel design and electrophoresis operating conditions were first optimized for reproducible analysis, after which two sieving matrices were tested. Finally, the method was validated using DNA samples isolated from intentionally infected milk.

Multiplex ligase‐based genotyping methods combined with CE

In this genomic era, the ability to assay multiple genomic hot spots that have strong clinical implications is greatly desired. Conventional PCR‐based methods suffer from frequent false‐positive detections, particularly when a multiplex analysis is desirable. As an alternative to the error‐prone conventional methods, multiplex ligase‐based genotyping methods combined with CE have a strong potential. In this review, both previously developed methods and emerging methods are described to reveal the specificity, sensitivity, and simplicity of the ligase‐based methods. For each step (ligation, amplification, and separation), the principles of several alternative methods are discussed along with their applications to explore the future development of ligase‐based diagnostic methods.

Precise expression profiling by stuffer-free multiplex ligation-dependent probe amplification.

In systems biological studies, precise expression profiling of functionally important gene sets is crucial. Real-time polymerase chain reaction is generally used for this purpose. Despite its widespread acceptance, however, this method is not suitable for multiplex analysis, resulting in an inefficient assay process. One alternative technology in the spotlight is multiplex ligation-dependent probe amplification (MLPA). But MLPA depends on length-based discrimination of amplified products, which complicates probe design and compromises analysis results. Here, we devised a variation of MLPA that utilizes conformation-sensitive capillary electrophoresis, and demonstrated the simplicity of the probe-design process and improved precision of the assay in analyses of 33 Escherichia coli metabolic genes and 16 Caenorhabditis elegans longevity-related genes. The results showed that relative expression could be quantitatively measured over a relevant dynamic range by using similar-sized probes. Importantly, the improved precision compared to conventional MLPA promises a wider application of this method for various biological systems.