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

Abstract

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.

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…But amplification efficiency varies between short and long stuffer sequences, so consistent PCR quantification is difficult to achieve (Chung and others ). Therefore, Shin and others () developed a stuffer‐free MLPA method to overcome the requirements for stuffer sequences. When stuffer‐free MLPA coupled with MCE (which used high‐resolution tri‐block copolymer‐poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) as the sieving gel) was used to detect contamination in infected milk, 5 pathogens ( V. parahaemolyticus , Y. enterocolitica , S. enteritidis , C. jejuni , and B. cereus ) were successfully detected and identified.…”

“…A schematic illustration of the CE‐SSCP analysis microdevice (Shin and others ). A 6‐cm‐long separation channel (from the cross‐junction to the anode reservoir) and a cross‐injector were filled with PEO‐PPO‐PEO triblock copolymer as a sieving matrix.…”

“…5)Consolidated criteria have begun to be established for integrating and comparing diagnostic results from different laboratories using different detection methods (Bogaerts and others ; Cheng and others ). 6)MPCR‐CE (Liao and others ; Patwardhan and others ) was given priority for development due to its high sensitivity, expeditiousness, high‐throughput, and cost‐effectiveness, while its drawbacks, such as multiple primer sets resulting in complex reaction conditions and some primer sets generating preferential PCR products (resulting in false results), were eliminated (Shin and others ; Shang and others ). 7)CE was combined with other techniques to capitalize on their advantages.…”

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Furthermore, they found that stuffer-free MLPA-CE-SSCP and Ab-MNPs together facilitated robust pathogen detection in various applications. To develop MLPA on a microchip platform, Shin et al [67] established MLPA-MCE-SSCP using a stuffer-free probe set for multiplex analysis of five foodborne pathogens and validated their method with DNA samples from infected milk (Figure 4).…”

Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009–2014)

“…There are 48 total Pluronic formulations; however, only a few are transparent and can be used in conjunction with DNA detection methods [ 161 ]. The Pluronic F108 matrix was utilized for the detection of pathogens [ 159 , 161 – 168 ] or human biomarkers [ 169 , 170 ].…”